System and method for microfluidic cell culture

ABSTRACT

Microfluidic devices and methods for perfusing a cell with perfusion fluid are provided herein, wherein the gravitational forces acting on the cell to keep the cell at or near a retainer or a retaining position exceed the hydrodynamic forces acting on the cell to move it toward an outlet. Also provided, are methods for assaying cell products within the microfluidic device.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of U.S. application Ser. No.13/178,395, filed Jul. 7, 2011, entitled “SYSTEM AND METHOD FORMICROFLUIDIC CELL CULTURE”, which application claims priority benefit ofU.S. Provisional Patent Application Ser. No. 61/362,213 filed on Jul. 7,2010, each of which is incorporated herein by reference in its entirety.

BACKGROUND

1. Field

This invention relates to microfluidic devices. In particular, theinvention relates to microfluidic devices and their uses and methods forculturing cells for extended periods of time.

2. Description of Related Art

Cell population heterogeneity poses a major obstacle to understandingcomplex processes that govern tissue-specific cellular responses,differentiation, and disease development. Averaged measurements of largenumbers of cells often obscure the variable responses of individual orrare cells. New technologies for studying cellular heterogeneity at thesingle cell level under well-defined chemical environments are thereforeof great interest in the study of cells (for example, stem cell fields).

The need for scalable single cell analysis is particularly acute in thestudy of hematopoietic stem cell (HSCs) growth and differentiation. Theanalyses of clonal cultures derived from single HSCs have been performedfor a number of years and these have already provided some insights intothe proliferation kinetics of the input cells, their in vitro responsesto varying growth factor conditions, and their rapid loss ex vivo of thedifferentiation pattern that is typically preserved when they expand invivo. Such experiments have shown that quiescence and delayed cell cycleentry correlate with higher potency (Brummendorf, T H. et al. J. Exp.Med. 188, 1117-1124 (1998); Audet, J. et al. Biotechnol. Bioeng. 80,393-404 (2002)), that asymmetric cell divisions are features of HSCswith long-term hematopoietic activity (Ma, N. N. et al. Biotechnologyand Bioengineering 80, 428-437 (2002)), and that the probability of HSCsexecuting a self-renewal decision in vitro is regulated by the types andconcentrations of growth factors to which it is exposed (Ma, N. N. etal. Biotechnology and Bioengineering 80, 428-437 (2002); Pineault, N. etal. Leukemia 19, 636-643 (2005); Pineault, N. et al. Molecular andCellular Biology 24, 1907-1917 (2004)). Recently, the study of HSCsusing automated time-lapse imaging and, in some cases, micropatternedsubstrates, has enabled increased time resolution and the identificationof new phenotypes associated with particular biological behaviors(Audet, J. et al. Biotechnol. Bioeng. 80, 393-404 (2002); El-Ali, J. etal. Nature 442, 403-411 (2006); Faley, S. L. et al. Lab Chip 9,2659-2664 (2009); Wang, Z. H. et al. Lab Chip 7, 740-745 (2007);Figallo, E. et al. Lab Chip 7, 710-719 (2007)). These latter approachesindicate the power of higher throughput micro-culture systems, eventhough they lack desirable features including variable schedules ofmedium exchange.

Integrated microfluidic systems provide many advantages for live-cellmicroscopy tracking studies. These advantages include low reagentconsumption, precise temporal control over growth conditions, and anability to work with but not be limited to small numbers of input cells.Although these advantages have been well explored to analyze yeast andbacterial cell responses (Balagadde, F. K. et al. Science 309, 137-140(2005); Taylor, R. J. et al. Proc. Natl. Acad. Sci. USA 106, 3758-3763(2009)), applications to mammalian cells are less developed. Whereasfluid- and cell-handling capabilities have been well established(El-Ali, J. et al. Nature 442, 403-411 (2006)), there have beenrelatively few reports of the application of programmable microfluidicsystems to the long-term analysis of biological responses presumablyowing to the difficulties in obtaining robust growth of mammalian cellsin microfluidic devices. Previous mammalian microfluidic culture systemshave been largely restricted to experiments with adherent cellsincubated for short periods of time (hours) (Faley, S. L. et al. LabChip 9, 2659-2664 (2009); Wang, Z. H. et al. Lab Chip 7, 740-745 (2007))in relatively large volumes of medium (Figallo, E. et al. Lab Chip 7,710-719 (2007)) and/or maintained under high perfusion rates (Kim, L. etal. Lab Chip 6, 394-406 (2006); Korin, N. et al. Biomed. Microdevices11, 87-94 (2009)). With a few notable exceptions (Lee, P. J. et al.Biotechnol. Bioeng. 94, 5-14 (2006); Hung, P. J. et al. Biotechnol.Bioeng. 89(1) (2005)), longer-term microfluidic mammalian cell culturehas been characterized by reduced growth rates and even deviations fromnormal phenotypes (Korin, N. et al. Biomed. Microdevices 11, 87-94(2009); Paguirigan, A. L. & Beebe, D. J. Integr. Biol. 1, 182-195(2009)). Technical hurdles in available devices include dehydration,immobilization of nonadherent cells to facilitate medium exchange andrecovery of the cultured cells for subsequent phenotypic or functionalanalysis. Furthermore, a microfluidic cell culture system that achievesculture conditions similar to those obtained in standard macrocultures,and allows for analysis of heterogeneous cell behaviour to generatedifferentiated cells both in vitro and in vivo would have practicalutility.

Microfluidic devices made of polydimethylsiloxane (PDMS), a transparentand biocompatible silicone elastomer, have been widely used forcell-culture applications and provide high gas permeability for theefficient exchange of oxygen and carbon dioxide. However, PDMS is alsopermeable to some small molecules (Berthier, E. et al. Lab Chip 8,852-859 (2008); Regehr, K. J. et al. Lab Chip 9, 2132-2139 (2009)) andallows for rapid transport of water vapor, which may result indehydration (Heo, Y. S. et al. Anal. Chem. 79, 1126-1134 (2007); Hansen,C. L. G. et al. J. Am. Chem. Soc. 12 8, 3142-3143 (2006)). The highsurface-to-volume ratios characteristic of nano-volume culture chambersfurther promote dehydration of microfluidic devices. In addition, smallhydrophobic molecules can diffuse in the elastomeric material and bedepleted from the medium. These variations may lead to spuriousbiological responses, reduced growth rates and even cell death.

SUMMARY

In a first embodiment, there is provided a method of culturing a cell,the method including: (a) retaining the cell at a retaining positionwithin a chamber having a chamber volume; and (b) flowing a perfusingfluid through the chamber, wherein the perfusing fluid enters thechamber through an inlet at an inlet position and exits the chamberthrough an outlet at an outlet position, wherein the perfusing fluid hasa greater velocity laminar flow adjacent the inlet and outlet positionsthan at the retaining position, and wherein a first region of thechamber is spaced apart from the retaining position and where the firstregion is interposed directly between the inlet and outlet positions.

In a further embodiment, there is provided a method of culturing a cell,the method including: (a) retaining the cell at a retaining positionwithin a chamber; (b) flowing a perfusion fluid into the chamber throughan inlet; and (c) flowing the perfusion fluid out of the chamber throughan outlet wherein the outlet is positioned such that gravitationalforces acting on the cell to keep it at or near the retaining positionexceed hydrodynamic forces acting on the cell to move it toward theoutlet.

In a further embodiment, there is provided a method of culturing a cell,the method including: retaining the cell in a volume of perfusion fluid,wherein the volume is less than about 10 mL; and placing the volume ofperfusion fluid in fluid communication with a reservoir fluid, whereinthe reservoir fluid has a volume greater than the volume of perfusionfluid. Alternatively, the perfusion fluid may be only in gaseouscommunication with the reservoir fluid.

The first region of the chamber may be defined as the volume of thechamber interposed directly between the inlet and outlet positions.Furthermore, the first region is spaced apart from the retainingposition such that the velocity of the perfusion fluid at the retainingposition is lower than the velocity of the perfusion fluid adjacent theinlet and outlet positions during perfusion. Similarly, the first regionis spaced apart from the retaining position such that the velocity ofthe perfusion fluid at the retaining position is lower than the velocityof the perfusion fluid within the first region during perfusion.Accordingly, the velocity of the perfusion fluid around a cell at theretaining position may be regulated such that the velocity of theperfusion fluid is lower at the retaining position than adjacent theinlet and outlet positions during perfusion. The velocity of theperfusion fluid around a cell at the retaining position may be regulatedsuch that the velocity of the perfusion fluid is lower at the retainingposition than the first region. The speed of the perfusion fluid may bedecreased to less than 50 μm/s as the perfusion fluid approaches theretaining position. The speed of the perfusion fluid may be decreased toless than 40 μm/s as the perfusion fluid approaches the retainingposition. The speed of the perfusion fluid may be decreased to less than30 μm/s as the perfusion fluid approaches the retaining position. Thespeed of the perfusion fluid may be decreased to less than 20 μm/s asthe perfusion fluid approaches the retaining position. The speed of theperfusion fluid may be decreased to less than 10 μm/s as the perfusionfluid approaches the retaining position. The speed of the perfusionfluid may be decreased to less than 5 μm/s as the perfusion fluidapproaches the retaining position. The speed of the perfusion fluid maybe decreased to less than 4 μm/s as the perfusion fluid approaches theretaining position. The speed of the perfusion fluid may be decreased toless than 3 μm/s as the perfusion fluid approaches the retainingposition. The speed of the perfusion fluid may be decreased to less than2 μm/s as the perfusion fluid approaches the retaining position. Thespeed of the perfusion fluid may be decreased to less than 1 μm/s as theperfusion fluid approaches the retaining position. The speed of theperfusion fluid may be decreased to 0 μm/s as the perfusion fluidapproaches the retaining position.

The chamber may have a top and a bottom, and the retaining position maybe at the bottom. The inlet position may be proximal to the top. Theoutlet position may be proximal to the top. The cell may be retained atthe bottom by gravitational forces. The method may further includeregulating osmolarity of the perfusion fluid within the chamber. Theregulating osmolarity of the perfusion fluid within the chamber mayinclude placing the chamber in fluid communication with a bathing fluid,wherein the bathing fluid has a volume greater than the chamber volume.The regulating osmolarity of the perfusion fluid within the chamber mayinclude placing the chamber in gaseous communication with a bathingfluid, wherein the bathing fluid has a volume greater than the chambervolume. The bathing fluid and the perfusion fluid may be iso-osmotic.The cell may be a suspension cell. The perfusion fluid may include anyone or more of: a cell culture medium; an immunostaining agent; anenzymatic reagent; a dye; an oil; and a bead-containing solution.

The length of the first region may be less than or equal to a length ofthe shortest distance between the retaining position and the firstregion. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be lessthan 3:1. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be lessthan 2:1. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be lessthan 3:2. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be lessthan 1:1. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 0.5. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 0.6. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 0.7. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 0.8. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 0.9. The ratio of the length of the first region to the shortestdistance between the retaining position and the first region may be morethan 1.0. The method may further include flowing the cell into thechamber prior to retaining the cell at the retaining position. Themethod may further include isolating a clone of the cell. The method mayfurther include tracking the progeny of the cell.

The flow of the perfusing fluid may be intermittent. The flow of theperfusing fluid may be continuous. The flow of the perfusing fluid maybe intermittent. The flowing of the perfusing fluid may be continuous.The replenishing of the perfusing fluid may be intermittent. Thereplenishing of the perfusing fluid may be continuous.

A value for x may be less than or equal to the value for y, wherein x isthe length of the shortest distance between the inlet and the outlet andy is the length of shortest distance between the retaining position aregion of the chamber that is interposed directly between the inlet andoutlet positions. The ratio of x:y of the chamber is greater than 0.5.

The method may further include replenishing the perfusion fluid. Theperfusion fluid and reservoir fluid may be iso-osmotic.

In a further embodiment, there is provided a microfluidic device forperfusing a cell with perfusion fluid, the device including: a chamber,having: (i) at least one inlet; (ii) at least one outlet; and (iii) acell retainer; wherein the inlet and the outlet are in fluidcommunication with the cell retainer, and wherein the outlet ispositioned such that, when the device is being perfused, gravitationalforces acting on the cell to keep it at or near the retainer exceedhydrodynamic forces acting on the cell to move it toward the outlet.

In a further embodiment, there is provided a microfluidic device forperfusing a cell with perfusion fluid, the device including: a chamber,having: (i) at least one inlet; (ii) at least one outlet; and (iii) acell retainer; wherein the inlet and the outlet are in fluidcommunication with the cell retainer; and wherein a first region of thechamber is interposed directly between the inlet and outlet positionsand is spaced apart from the cell retainer.

The microfluidic device may further include an osmolarity regulator forregulating the osmolarity of the perfusion fluid. The osmolarityregulator may include an iso-osmotic reservoir in fluid communicationwith the chamber. The microfluidic device may further include areservoir for holding a reservoir fluid, wherein the reservoir is influid communication with the chamber. The reservoir fluid may beiso-osmotic with the perfusion fluid. The microfluidic device mayfurther include flow channels in fluid communication with the chambervia the at least one inlet and the at least one outlet. The chamber mayhave a top and a bottom, and the cell retainer is at the bottom. Theinlet may be proximal to the top. The outlet may be proximal to the top.The cell may be a suspension cell. The perfusion fluid may include anyone or more of: a cell culture medium; an immunostaining agent; anenzymatic reagent; a dye; an oil; and a bead-containing medium. Thedistance between the inlet and the outlet may be less than a distancebetween the cell retainer and the outlet. The ratio of the length of thedistance between the inlet and the outlet to the distance between thecell retainer and the outlet may be less than 2:1. The ratio of thelength of the first region to the shortest distance between theretaining position and the first region may be less than 3:1. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be less than 2:1. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be less than 3:2. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be less than 1:1. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 0.5. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 0.6. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 0.7. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 0.8. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 0.9. The ratioof the length of the first region to the shortest distance between theretaining position and the first region may be more than 1.0.

The device may be an array of at least 100 chambers. The chambers may beconnected in parallel. The chambers may be serially connected. Thechambers may be connected in parallel and in series. The chambers may beconnected partially in parallel and partially in series. Themicrofluidic device may be operable to be perfused intermittently. Themicrofluidic device may be operable to be perfused continuously.

In a further embodiment, there is provided a use of a microfluidicdevice described herein for tracking progeny of the cell.

In a further embodiment, there is provided a use of a microfluidicdevice described herein for selection of a clone of the cell.

In a further embodiment, there is provided a use of a microfluidicdevice described herein for observing cell-cell interactions.

In a further embodiment, there is provided a use of a microfluidicdevice described herein for observing autocrine effects of the cell.

The selection may be based on the amount of a factor produced by thecell or the clone. The factor may be a protein. The factor may be anucleic acid. The cell may be a suspension cell. The use may furtherinclude recovery of the cell or clone thereof.

In a further embodiment, there is provided a method, the methodincluding: (a) retaining a cell at a retaining position within amicrofluidic chamber having a chamber volume; (b) flowing a perfusingfluid through the microfluidic chamber, wherein the perfusing fluidenters the chamber through an inlet at an inlet position and exits thechamber through an outlet at an outlet position; and (c) measuring acell product produced by the cell within the microfluidic chamber;wherein the perfusing fluid has a greater velocity laminar flow adjacentthe inlet and outlet positions than at the retaining position, andwherein a first region of the chamber is spaced apart from the retainingposition, wherein the first region is interposed directly between theinlet and outlet positions.

In a further embodiment, there is provided a method, the method,including: (a) retaining a cell at a retaining position within amicrofluidic chamber; (b) flowing a perfusion fluid into themicrofluidic chamber through an inlet; (c) flowing the perfusion fluidout of the microfluidic chamber through an outlet wherein the outlet ispositioned such that gravitational forces acting on the cell to keep itat or near the retaining position exceed hydrodynamic forces acting onthe cell to move it toward the outlet; and (d) measuring a cell productproduced by the cell within the microfluidic chamber.

In a further embodiment, there is provided a method, including:retaining a cell in a volume of perfusion fluid, wherein the volume isless than about 10 nL; placing the volume of perfusion fluid in fluidcommunication with a reservoir fluid, wherein the reservoir fluid has avolume greater than the volume of perfusion fluid; and measuring a cellproduct produced by the cell within the perfusion fluid.

The measuring the cell product may be selected from one or more of:lineage staining; cell-surface protein staining; antibody staining;enzymatic assay; RT-PCR analysis; PCR analysis; sequencing; functionalassay; and bead capture assay to characterize the cells. The method mayfurther include selecting cell clones based on cell characteristics. Thecell characteristics may be selected from one or more of: quantity ofsecreted product, quality of secreted product, proliferation,morphology, gene expression, fluorescent reporter, surface proteins,genealogical pedigree, viability, apoptosis, autophagy, metabolism,clone homogeneity, and clone heterogeneity.

In a further embodiment, there is provided a microfluidic device forperfusing a cell with perfusion fluid, the device including: a chamber,having: (i) at least one inlet; (ii) at least one outlet; and (iii) acell retainer; wherein the inlet and the outlet are in fluidcommunication with the cell retainer, and wherein the outlet ispositioned such that, when the device is being perfused, gravitationalforces acting on the cell to keep it at or near the retainer exceedhydrodynamic forces acting on the cell to move it toward the outlet.

In a further embodiment, there is provided a microfluidic device forperfusing a cell with perfusion fluid, the device including: a chamber,having: (i) at least one inlet; (ii) at least one outlet; and (iii) acell retainer; wherein the inlet and the outlet are in fluidcommunication with the cell retainer; and wherein a first region of thechamber is interposed directly between the inlet and outlet positionsand is spaced apart from the cell retainer.

The microfluidic device may further include a perfusion fluid comprisingone or more of the following: an immunostaining agent; an enzymaticreagent; a dye; and a functionalized bead. The immunostaining agent maybe selected from one or more of: monoclonal antibodies; polyclonalantibodies; fluorophores; and blocking solution. The enzymatic reagentmay be selected from one or more of: horseradish peroxidase; andluminal. The dye may be selected from one or more of: rhodamine;fluorescein; calcein; Hoescht; Trypan blue; propidium iodide; and Giemsasolution. The functionalized bead may be selected from one or more of:magnetic beads; polymer beads; protein A-coated beads; and proteinG-coated beads.

In a further embodiment, there is provided a method of culturing a celland measuring cell products within a microfluidic chamber, the methodincluding: (a) retaining the cell within the microfluidic chamber at aconcentration of ≧10,000 cells per mL; (b) flowing a perfusing fluidthrough the microfluidic chamber; and (c) measuring a cell productproduced by the cell within the microfluidic chamber.

The cell product may be secreted by the cell. The cell product may bequantified directly or indirectly by an intracellular fluorescentreporter. The cell product may be intracellular and may be released bycell lysis prior to characterization of the product. The measuring thecell product may be selected from one or more of: lineage staining;cell-surface protein staining; antibody staining; enzymatic assay;RT-PCR analysis; PCR analysis; sequencing; functional assay; and beadcapture assay to characterize the cells. The method may further includeselecting cell clones based on cell characteristics. The cellcharacteristics may be selected from one or more of: quantity ofsecreted product, quality of secreted product, proliferation,morphology, gene expression, fluorescent reporter, surface proteins,genealogical pedigree, viability, apoptosis, autophagy, metabolism,clone homogeneity, and clone heterogeneity. The measuring of the cellproduct may be selected from one or more of the following: antibodystaining; enzymatic assaying; functional assaying, surface capturing, orbead capturing. The cell or clone may be maintained under cultureconditions that allow subsequent clonal expansion. The cell within themicrofluidic chamber may be at a concentration ≧20,000 cells per mL. Thecell within the microfluidic chamber may be at a concentration ≧30,000cells per mL. The cell within the microfluidic chamber may be at aconcentration ≧40,000 cells per mL. The cell within the microfluidicchamber may be at a concentration ≧50,000 cells per mL. The cell withinthe microfluidic chamber may be at a concentration ≧60,000 cells per mL.The cell within the microfluidic chamber may be at a concentration≧70,000 cells per mL. The cell within the microfluidic chamber may be ata concentration ≧80,000 cells per mL. The cell within the microfluidicchamber may be at a concentration ≧90,000 cells per mL. The cell withinthe microfluidic chamber may be at a concentration ≧100,000 cells permL. The cell within the microfluidic chamber may be at a concentration≧125,000 cells per mL. The cell within the microfluidic chamber may beat a concentration ≧250,000 cells per mL. However, the upper limit forcell density may be as high as 1 E9 cells/mL. The measuring of thesecretion product may occurs between 5 minutes and 6 hours from theretaining of the cell within the microfluidic chamber.

Furthermore, the device may be selected based on the cell concentrationsto avoid the need to dilute or concentrate the cells in solution, whichmay damage the cells, whereby the selection of chamber sizes is possibleto promote a particular seeding density. For example, with a more dilutecell solution, a larger chambers may be selected and with a moreconcentrated cell solution, a smaller chamber may be selected.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In drawings which illustrate embodiments of the invention.

FIG. 1 is a top view of a microfluidic device with two expanded views(11×12 chamber view (size reference bar=1 mm) and a 4 chamber view (sizereference bar=100 μm)) to magnify details of the microfluidic deviceaccording to an embodiment.

FIG. 2 is an exploded oblique view of a portion of the microfluidicdevice depicted in FIG. 1, showing the various layers associated with anindividual microfluidic device.

FIG. 3 is an oblique view from below of 4 microfluidic chambers depictedin FIG. 1 with associated fluid channels and control layers.

FIG. 4 is a cross-sectional view of a chamber and a channel of themicrofluidic device depicted in FIG. 1, showing the dimensions andvolume of the chamber, and a depiction of fluid speed in the controllayer and chamber while the chamber is being perfused.

FIG. 5 is an oblique view of the microfluidic device (array) depicted inFIG. 1, further showing the microfluidic device containing aniso-osmotic bath pressurized by a syringe, the inlet and outlet, and thecontrol lines (pumps and valves) which may be connected to solenoidactuators (not shown).

FIG. 6 shows differences in cell survival during microfluidic culture inthe indicated conditions compared to the high [SF] condition, whereinthe cells were imaged every 12 min, and survival curves were normalizedto a third-order polynomial fit for the high [SF] conditions.

FIG. 7 shows cumulative division kinetics of primary HSCs that arecycling (excluding dead and quiescent cells) in the indicated in vitroconditions for the first and second divisions.

FIG. 8 shows individual growth curves of primary murine HSCs underdifferent Steel factor (SF) exposure conditions, where the growth curveswere generated using the enhanced bifocal image analysis algorithm andthe analysis was started after 21 hours to allow small quiescent cellsto reach a suitable size for detection by image analysis.

FIG. 9 a is a scatter plot depicting cell counts before and after mediumexchange (2 μL/min for 10 minutes) showing minor variations attributableto cell division and cell death.

FIG. 9 b is a graph depicting the efficiency of cell recovery fromchambers by cell count in individual chambers and recounting of cellssuccessfully transferred to a well, showing that on average 91% of thecells from individual colonies of different sizes could be recovered.

FIG. 9 c is a graph depicting lineage tracking of cells for 3 clonesfollowing manual inspection of images, where the cells were imaged every5 minutes and media was exchanged every 6 hrs.

FIG. 10 is a graph comparing the of average growth rates of ND13 cellsin 24-well dish culture, single cell 96-well culture, a microfluidicdevice as described herein with and without an iso-osmotic reservoir(arrows show growth medium exchange).

FIG. 11 is a graph of cell concentration (1 cell, 2-15 cells, 16-30cells, 31-45 cells, and >45 cells) over time for various seedingdensities grown in a microfluidic device according to an embodiment, butwith no medium exchange and no iso-osmotic bath. This Figure shows astrong inverse correlation with the initial number of cells in eachchamber.

FIG. 12 a are time-lapse images of clonal expansion in a chamber of amicrofluidic device according to an embodiment (at 0 hr and at 6 hrintervals thereafter until 66 hrs.).

FIG. 12 b is a histogram depicting distribution of average doublingtimes of clonal ND13 cultures up to 72 hrs. in a microfluidic deviceaccording to an embodiment, showing that the cells growth rates arehighly heterogeneous, whereby only a small fraction of fast growingcells contributed to the overall growth rate, while 52% of the cells didnot give rise to colonies (i.e. cells marked ‘no proliferation’ (52%)did not divide during this period or died).

FIG. 12 c is a graph depicting the proliferation profile of individualclones over time (as counted by automated image analysis for individualND13 cells) in a microfluidic device according to an embodiment, alsoshowing highly variable growth rates.

FIG. 13 a is a graph depicting the proliferation of lin⁺ clones in ND13cells grown in a microfluidic device according to an embodiment, whereinthe majority of the lin⁺ cells either died or did not give rise tocolonies.

FIG. 13 b is a graph depicting the proliferation of lin⁻ clones in amicrofluidic device according to an embodiment, wherein the lin⁻ cellsgave rise to colonies of different sizes.

FIG. 13 c is an image depicting live immunostaining of small clonalpopulations in a microfluidic device according to an embodiment, whereinNup98-HOXD13 clones were stained for lineage markers B220, Gr-1, andMac-1 at 0 Hrs. and after 72 hours inside the microfluidic device.

FIG. 13 d is a histogram depicting the progeny of Nup98-HOXD13 clonestaken from a microfluidic device according to an embodiment and acontrol plate to compare colony forming cells (CFC—colonies/100 cells)using methylcellulose assays.

FIG. 14 a is a schematic diagram of a study to compare primaryhematopoietic stem cells (HSC) activity in NUP98-HOXA10hd(NA10dh)-transduced hematopoietic populations cultured in a microfluidicdevice according to an embodiment as compared to hematopoieticpopulations cultured in a macroscale 96-well plate as compared bycompetitive repopulating cell (CRU) assay.

FIG. 14 b shows a comparison of macroscale 96-well plate cultured cells(control) with microarray cultured cells, whereby NUP98-HOXA10hd cellsmaintained functional HSC activity after being cultured in themicrofluidic array according to an embodiment and were able toreconstitute the blood-forming system of lethally irradiated mice.

FIG. 15 a is a histogram depicting the ability of NA10 hd hematopoieticpopulations cultured in a microfluidic device according to an embodimentand hematopoietic populations cultured in a macroscale 96-well plate toproduce myeloid and lymphoid lineages as defined by lineage markersGr-1/Mac-1, B220, and CD4/CD8.

FIG. 15 b is a histogram depicting distribution of average doublingtimes of single HSC cells (NA10hd) in a microfluidic device according toan embodiment after being transduced with NA10 hd (apparent doublingtime 13 hrs).

FIG. 16 shows a microfluidic cell culture array for temporal stimulationand parallelization of experiments, wherein the microfluidic cellculture array contains 6,144 chambers and can support up to 8 differentconditions simultaneously, but only the top half of the array was usedto study murine HSCs due to the relatively small cell numbers and 6different conditions were distributed across the array as shown.

FIG. 17 a shows representative images from an automated image analysisalgorithm for cell quantification of cells in a chamber of amicrofluidic device according to an embodiment, where segmentation wasaccomplished through three main steps: chamber segmentation (A-E),cell-containing region segmentation (F-J), and then single cellisolation (K-O). First, the individual chambers are segmented from theimage background.

FIG. 17 b shows a plot comparing automated image analysis depicted inFIG. 17 a and manual cell counts, wherein the straight line representsthe 1:1 slope.

FIG. 18 shows a mature myeloid population derived from lineage negativeND13 cells, where the ND13 cells were stained for Gr-1, Mac-1 and B220and sorted by flow cytometry and the lin⁻ fraction was cultured for 9days and gave rise to a new lin⁺ population.

FIG. 19 is a graph of average fluorescence intensity as a function ofperfused volume.

FIG. 20 a is a scatter plot comparing the results of manual cell countsand automated cell counts employing a bifocal algorithm

FIG. 20 b is a histogram showing absolute differences between thealgorithm and manual counts.

FIG. 21 shows a percentage of dividing cells over time for mouse HSCscultured in the microfluidic array under high SF concentration (300 ngml-1), where single cells were imaged every 4 min, and the times for thefirst, second and third divisions were identified by manual inspectionsof the videos for 46 cells.

FIG. 22 shows typical seeding density of around 25% of the 1,600chambers contained single cells at a loading concentration of 2×10⁶cells ml⁻¹ (total: 1,034 cells), close to the expected Poissondistribution and the theoretical maximum of a Poisson distribution foran average of 1 cell per well (total: 1,600 cells).

FIG. 23 shows the bead immunocapture assay to measure antibody secretionfrom single cells in 1,600 chambers, wherein (a) Cells were loadedstochastically into an array of 4.1 nl chambers. (b) Protein A-coatedbeads (diameter: 4.9 μm) were introduced and (c) the chambers wereisolated for 2 h using microvalves. (d) The array was then washed and(e) Alexa 594-Rabbit anti-human IgG (H+L) F(ab′)₂ fragment wasintroduced for 15 min. (f) The array was washed again and fluorescentimages were taken to identify producing cells. (g) Example offluorescent and bright field images from the bead immunocapture assay(top left) followed by time-lapse imaging of the clone for 4.5 days. Thepolystyrene beads are the darker objects (black arrow) while the cellsare more transparent (white arrow). Cloning medium was used throughoutthis assay. Scale bar, 100 μm.

FIG. 24 shows antibody selection for the bead immunocapture assay,wherein fluorescence intensity was compared for beads exposed to cloningmedium only (non-specific binding) and supernatant from a 3-day batchculture of CHO cells (antibody signal). The following labeled antibodieswere tested: (1) Dylight 594-conjugated F(ab′)₂ fragment of rabbitanti-human IgG (H+L), (2) Dylight 594-conjugated F(ab′)₂ fragment ofgoat anti-human IgG (H+L), (3) Dylight 594-conjugated F(ab′)₂ fragmentof donkey anti-human IgG (H+L), (4) Dylight 594-conjugated F(ab′)₂fragment of goat anti-human IgG, F(ab′)₂ fragment-specific, (5) Alexa594-conjugated goat anti-human IgG (H+L), (6) Biotin-conjugated F(ab′)₂fragment of chicken (H+L) labeled with Dylight 594-nutravidinand and (7)FITC-conjugated F(ab′)₂ fragment of chicken (H+L). Lines 8 and 9 showbead autofluorescence in the red and green channels respectively. TheDylight 594-conjugated F(ab′)₂ fragment of rabbit anti-human IgG (H+L)had the highest signal-to-noise ratio and was chosen for the assay.

FIG. 25 shows automated bead segmentation and measurement of mean beadintensity (a) The bright field image was first used to locate the wellcontours using blurring and subtraction of the original image. (b) Thewell contour was filled to create a mask of the well. (c) The brightfield image was segmented using a set threshold and (d) beads werefilled by a combination of dilation and erosion. (e) Both images (b) and(d) were multiplied to obtain a bead mask. (f) The bead mask in (e) wasused to segment the beads and obtain the total bead area. (g) Thefluorescence image was multiplied by the bead mask in (e), resulting in(h) the measurement of the total fluorescence intensity. The mean beadintensity was measured by dividing the total intensity by the total beadarea.

FIG. 26 shows a typical bead distribution (a) typical bead distributionin the microfluidic cell culture array, where on average, 129 beads perchamber were loaded using a solution containing 2 mg beads ml⁻¹.

FIG. 27 shows the accuracy of the bead intensity measurement algorithmwith images manually curated to correct for segmentation errors andcompared to the automated measurement. The automated image analysisalgorithm accurately measured bead mean fluorescence intensity.

FIG. 28 shows a mean bead intensity as a function of the antibodyconcentration, wherein the antibody solutions were mixed to constantamounts of beads in tubes (0.5 mg ml⁻¹), incubated for 2 h, washed, andincubated for 15 min with the labeled antibody. After multiple washes,the beads were mounted on glass slides and bright field and fluorescentpicture sets were taken to measure the mean bead intensity associatedwith antibody to bead ratios. The red line represents the data fit to aLangmuir equation (I_(max)=3592; K=1.50 ml μg⁻¹). Typical meanintensities for chambers containing the top 5% producer cells (shadedarea) fell below saturation levels. Saturation occurred at around 4 μgml⁻¹, corresponding to or 8 μg antibody (mg bead)⁻¹ (dashed line),consistent with the manufacturer specifications.

FIG. 29 shows a quantification of antibody secretion The mean beadintensity distinguishes high producer cells from non-producer cells byseveral orders of magnitude. Analysis of wells that did not containcells (n=848 chambers) compared to chambers that contained single cells(n=397 chambers) showed minimal cross-contamination between chambers.Empty wells had a distribution of mean bead intensities (μ=2.96;CV=3.58) comparable to the distribution of non-producer cells (μ=2.70;CV=3.17). 32.7% of single cells had levels of mAb above the intensity ofempty wells.

FIG. 30 shows an improved cell growth and cloning efficiency in themicrofluidic cell culture array (a) Growth curves (error bars, s.d.) ofCHO cells cultured in shake flasks (n=3 experiments in triplicate seededat 2.5×10⁵ cells ml⁻¹), and as single cells in 96 well plates (n=3experiments; 27-36 clones per plate) or in microfluidic array (n=3experiments; 50 clones tracked per experiment) (b) Improved cloningefficiency in the microfluidic cell culture array (n=3 experiments;error bars, s.d.; P value=0.06). The cloning efficiency was measured asthe percentage of clones that had more than 8 cells at 72 h. Thiscriterion was selected based on the doubling rate of clones growing inmultiwell plates (23.2 h) from (a) as clones growing at a normal rateshould have undergone at least 3 divisions by 72 h.

FIG. 31 shows simultaneous measurement of membrane-bound antibody andantibody secretion (a) Example of a secreting cell with no antibodybound on its membrane. (b) Example of a non-secreting cell withmembrane-bound antibody. Cell outlines are highlighted in green. Scalebar, 100 μm. (c) Membrane-bound antibody shows poor correlation withsecretion levels.

FIG. 32 shows a recovery of selected clones, with an example of a CHOcell cultured in the microfluidic array for 5 days and recovered in a96-well plate for further expansion.

FIG. 33 shows a selection of high-producer clones (a) Comparison ofmicrofluidic secretion assay (red) with titers from at the 24-well platestage (blue). Single cells from one experiment (n=308) are ranked basedon the mean fluorescence bead intensity in each microfluidic chambers(red). Out of these, 60 clones were recovered and scaled up to the24-well plate stage (blue). (b) Out of the 10 of the top 12 clones thatwere scaled up, 4 clones were already showing signs of decreasedproductivity at the 24-well plate stage (shaded area) and were excludedfrom the screen. (c) Batch shake flask titers of the remaining 6 clonesare shown after 3, 5 and 7 days in culture (n=2; error bars, s.d.). (d)The maximum cell specific productivity measured after 5 days in batchshake flask culture is shown as a function of the microfluidic meanfluorescence intensity measured on the single cell that gave rise to theclone (n=2; error bars, s.d.).

FIG. 34 shows batch shake flask titers of eliminated clones with clonesthat scored high in the microfluidic assay but showed drops inproductivity at the 24-well plate stage (a) Titers after 3, 5 and 7 daysin culture (n=2 flasks; error bar, s.d.) and (b) cell specificproductivities (n=2 flasks; error bar, s.d.) at day 5 are presented.

FIG. 35 shows an analysis of intraclonal heterogeneity (a) Thetop-ranked clone was analyzed in the microfluidic device using the beadimmunocapture assay. Single cell analysis revealed a much tighterdistribution for the clone than from the cell pool from which itoriginated. (b) All wells were analyzed and averaged based on the numberof cells they contained. There was a linear correlation between the meanbead intensity and the number of cells for up to 3 cells (R²=0.99).

DETAILED DESCRIPTION Definitions

A “microfluidic device”, as used herein, refers to any device thatallows for the precise control and manipulation of fluids that aregeometrically constrained to structures in which at least one dimension(width, length, height) may be less than 1 mm.

“Perfusion” or “perfusing”, as used herein, refers to the passage offluid, such as culture media, over cells for the purposes of nutritivedelivery and waste removal. A person skilled in the art will understandthat the fluid does not necessarily flow over the cell, but mayultimately arrive at a cell by the process of diffusion. Furthermore,perfusion or perfusing of a chamber or the microfluidic device as awhole may be continual or intermittent provided that the fluid exchangeprovides sufficient nutrient delivery and/or waste removal and or otherfactors or reagents to keep the cells viable (if that is the desiredresult) and/or to maintain desired conditions for the particular celland/or assay as desired.

A “perfusion fluid”, as used herein, refers to any fluid with which acell in a chamber is perfused. A person skilled in the art willunderstand that a perfusion fluid may comprise factors or reagents withwhich it is desired to present to the cell. Factors or reagents mayinclude components of culture medium (e.g. sugars, proteins, aminoacids, vitamins, salts), proteins (e.g. interferon-α, TAT, fibronectin,bovine serum albumin), small molecules (e.g. all-trans retinoic acid,imatinib), growth factors (e.g. IL-3, IL-6, IL-11, SCF, GM-CSF),immunostaining agents (e.g. monoclonal antibodies, polyclonalantibodies, fluorophores, blocking solution), enzymatic reagents (e.g.horseradish peroxidase, luminol), oils (e.g. mineral oil, fluorinatedoil), dyes (e.g. rhodamine, fluorescein, Hoescht, functionalized beads(e.g., magnetic beads, polymer beads, protein A-coated beads, proteinG-coated beads), buffers (e.g. PBS, Hank's balanced solution, HEPES),PCR solutions (e.g. polymerase, nucleid acids, primers), celltransfection solutions (e.g. fibronectin, retronectin,polyethylenimine), cell fixation solutions (e.g. ethanol, formaldehyde),miRNAs, siRNAs, molecular beacons, amino acids (e.g. glutamine),antigens, semi-solid matrix (e.g. methylcellulose, Matrigel®), etc.Alternatively, a perfusion fluid may include a lysis solution to lysethe cells and allow for the assay of intracellular products.

A “chamber” or “cell capture chamber”, as used herein, refers to anenclosed space within a microfluidic device in which one or more cellsmay be isolated from a larger population of cells as the cells areflowed through the device. Each chamber will have at least one inlet forpermitting fluid, including fluid containing cells, to enter thechamber, and at least one outlet to permit fluid to exit the chamber.Persons skilled in the art will understand that an inlet or an outletcan vary considerably in terms of structure and dimension, and may bereversibly switched between an open position, to permit fluid to flowinto or out of the chamber, and a closed position to seal the chamberand thereby isolate and retain its contents, whereby the aperture mayalso be intermediate between the open and closed positions to allow somefluid flow. Each chamber will further have at least one cell retainingposition which may comprise at least one cell retainer.

The direction of fluid flow through the chamber dictates an “upstream”and a “downstream” orientation of the chamber. Accordingly, an inletwill be located at an upstream position of the chamber, and an outletwill be generally located at a downstream position of the chamber. Itwill be appreciated by a person of skill in the art, that thedesignation of an “inlet” or an “outlet” may be changed by reversing theflow within the device or by opening one or more alternativeaperture(s).

A “cell retaining position” or “retainer position”, as used herein,refers to a location in the chamber at which a cell is maintained duringcell culture and media exchange. A retaining position may include atleast one cell retainer. According to some embodiments, the retainingposition may be a determined position within the chamber. However, aperson skilled in the art will understand that a retaining position maycomprise a zone within the chamber. The important characteristic of aretaining position is that hydrodynamic forces are insufficient tofacilitate the escape of a cell through the outlet while the cell is inthe retaining position and shear forces on the cell, if any, do notdamage the cell. Depending on the cell type, the cell may adhere, eitherweakly or strongly, to the cell retaining position or a cell retainer ormay be held in place by gravitational forces or a cell trap.

A “cell retainer”, as used herein, refers to any structure which servesto maintain a cell within a retaining position. In a simple embodiment,the cell retainer may be the bottom of the chamber and the cells may beheld in place by gravitational forces. Alternatively, a cell retainermay include a cell trap positioned to receive (and retain) a cell thatis flowed into the chamber. Furthermore, a substrate may be provided atthe cell retaining position that facilitates retention of the cells. Forexample, extracellular matrix (ECM) components or integrin orfunctionalized beads etc. may be deposited on at the retaining positionto facilitate retention of cells.

An “inlet” or an “outlet”, as used herein, may include any aperturewhereby fluid flow is restricted through the inlet or outlet. There maybe one or more valves to control flow, or flow may be controlled byseparating the fluid channels, which lead to the inlets and outlets witha layer which prevents flow (for example, a control layer or isolationlayer). Alternatively, flow may be regulated by the rate at which passedthrough the device. An “inlet position”, as used herein, refers to aposition in the chamber where an inlet is located. Similarly, an “outletposition”, as used herein, refers to a position within a chamber wherean outlet is located. According to embodiments, the inlet position,outlet position, and retaining position will not be co-linear.

A “first region”, as used herein, refers to a region of the chamber thatis interposed directly between the inlet and outlet positions. In someembodiments the first region is spaced apart from the retainingposition. According to some embodiments described herein, the firstregion may be generally at the top of the chamber and the cell retainingposition may be generally at the bottom of the chamber, such that thevelocity of the fluid in the cell retaining position is slower than thevelocity of the fluid in and around. In some embodiments the velocity offluid in cell retaining position is or approaches zero and the onlyperfusion fluid that enters the cell retaining position is by diffusionor convection, thereby providing fresh diffusion fluids to the cell.According to some embodiments described herein, the speed of the fluidflow in the first region will be sufficiently low such that thehydrodynamic forces of the fluid urging a cell from the retainingposition to an outlet are exceeded by forces, e.g. gravitational forces,urging the cell toward the cell retaining position.

A “cell trap”, as used herein, refers generally to a means for receivingand retaining cells at a pre-determined location over time. A cell trapmay comprise localized surface modifications for chemical immobilizationof a cell. Alternatively, the cell trap may be a mechanical trap, ahydrodynamic trap (Skelley, A M et al. Nat Methods 6(2):147-152 (2009);Li, P.C. H. et al. Lab on a Chip 4, 174-180 (2004); Li, X. & Li, P. C.H. On-Chip Dye Loading, Cell Contraction by Chemical Stimulation, andQuantitative Fluorescent Analysis of Intracellular Calcium. Anal. Chem.77, 4315-4322, doi:10.1021/ac048240a (2005); Di Carlo, D. et al. Anal.Chem. 78, 4925-4930, doi:10.1021/ac060541s (2006)), a hydrodynamicbalancing trap (Rowat, A. C. et al. Proceedings of the National Academyof Sciences 106, 18149-18154, doi:10.1073/pnas.0903163106 (2009); andKobel, S. et al. Lab on a Chip 10, 857-863 (2010)), an active valvingtrap (Warren L, et al. Proc Natl Acad Sci USA 103(47):17807-17812(2006); Skelley, A M et al. Nat Methods 6(2):147-152 (2009); Li, P. C.H. et al. Lab on a Chip 4, 174-180 (2004); King, K. R. et al. Lab on aChip 7, 77-85 (2007); Marcy, Y. et al. Proc. Natl. Acad. Sci. U.S.A.104, 11889-11894 (2007)), a dielectrophoretic trap (Voldman, J. et al.Anal. Chem. 74, 3984-3990, doi:10.1021/ac0256235 (2002)), a DNAimmobilization trap (Toriello N M, et al. Proc Natl Acad Sci USA105(51):20173-20178 (2008)), a gel encapsulation trap (Braschler, T. etal. Lab on a Chip 5, 553-559 (2005)), a magnetic trap, an acoustic trapor an optical trap (Neuman, K. C. et al. Biophys. J. 77, 2856-2863(1999)). In various embodiments described herein, a cell trap willgenerally be positioned directly in the path of the smaller crosssectional of cell flow created by the funnel. Where a mechanical funnelis used according to various embodiments described herein, a trap may bepositioned directly after the downstream opening of the funnel.Furthermore, additional cell trapping and funneling methods may be foundin PCT/CA2011/000612.

A “mechanical trap”, as used herein, refers to a physical cell trap suchas a cage.

A “hydrodynamic trap”, as used herein, refers to a cell trap in whichthe force of the fluid in motion plays a role in retaining a trappedcell in its position. A hydrodynamic trap may be also be comprised of amechanical trap in which a cell is captured and retained. Exemplarymechanical traps are described in PCT/CA2011/000612. In certainembodiments hydrodynamic traps may be utilized. However, it may bedesirable to have three or more inlets to the cell capture chamber sothat the flows may be adjusted in order to direct cells to the traps.

A “dielectrophoretic trap”, as used herein, refers to a cell trap inwhich cells, being dielectric objects, are retained by the forcesgenerated by a non-uniform electric field.

A “magnetic trap”, as used herein, refers to a cell trap employingmagnetic fields to retain cells. Typically, cells will be labeled withmagnetic particles, and then positioned and retained by the magneticfields. However, magnetic traps can also be used to trap-non-magneticcells in suitable buffers.

An “acoustic trap”, as used herein, refers to a cell trap in whichultrasonic standing waves are used to generate stationary pressuregradients that exert forces that position and retain cells.

An “optical trap”, as used herein, refers to a cell trap in which atightly focused laser beam, typically a near-infra red laser beam, isused to draw cells in the direction of the beam.

The size of the cell trap may be varied according to the size, type,mechanical properties, or number of cells that are desired to betrapped. A microfluidic device according to various embodiments mayfurther include a combination of trap designs for the capture of a rangeof cell types. Furthermore, each chamber could include multiple traps oreach chamber or a subset of chambers may be optimized to capture aparticular cell type. In such embodiments, the frequency of cells of aparticular size or having particular characteristics that are trappedmay be used for diagnostic or other assay purposes. Alternatively, thecontents of a group of cells caught in a single trap may be processedand analyzed.

A chamber may further include cell funnel, and a “cell funnel” as usedherein, refers to an apparatus which is designed to focus the flow ofcells from a first location, where the cells are dispersed, to one ormore desired second or more locations within the chamber wherein thecell funnel has a smaller cross sectional area of cell flow. The cellfunnel may exert a force to direct cells towards the one or more desiredlocations within the cell capture chamber. For the purposes of clarity,“force” is defined herein as any influence that causes a free body (e.g.a cell) to undergo a change in velocity. Funnels may either span theentire height and/or width of the cell capture chamber, or partiallyspan the height and/or width. Exemplary cell funnels are described inPCT/CA2011/000612.

A “bathing fluid”, as used herein, refers to any fluid which is used toregulate the osmolarity of fluid, e.g. perfusion fluid, within achamber. A bathing fluid may be iso-osmotic with the perfusion fluid orsufficiently close to iso-osmotic such that the osmolarity of the fluidremains in a range that is suitable for cell culturing. According to anembodiment described herein, bathing fluid will generally be present ina volume greater than a chamber. The bathing fluid may be a in areservoir that is in gaseous communication with the chamber. Forexample, the reservoir may be separated from the chamber by agas-permeable PDMS membrane, wherein the water exchange occurs in vaporphase.

An “osmolarity regulator” is a system for regulating the osmolarity ofthe perfusion fluid within a chamber and/or a microfluidic device as awhole. For example, the osmolarity regulator may comprise an iso-osmoticreservoir or bath in fluid communication with the chamber, wherein theiso-osmotic reservoir is filled with or resupplied with a bathing fluidthat may be iso-osmotic with the perfusion fluid. The term bath andreservoir are used interchangeably with regards to osmolarity regulationof the device.

Alternatively, osmolarity may be regulated in the chambers by immersingthe chambers in a large volume media bath that would be recirculated tomaintain osmolarity. Alternatively, the osmolarity may be regulated inthe chambers by enclosing the microfluidic device in an environmentallyregulated enclosure.

“Aspect ratio”, as used herein, refers to the ratio (y:x) of theshortest distance between the cell retaining position and the firstregion (y) to the length of the first region (x). In various embodimentswhere the inlet and outlet is at the top of the chamber, such that thefirst region is horizontal and defines an area that is interposeddirectly between the inlet and outlet positions, the aspect ratio willeffectively been the ratio of the height of the chamber (minus theheight of the first region) to the width of the chamber.

A “hydrodynamic force”, as used herein, refers to a force exerted by afluid in motion.

An “adherent cell”, as used herein, refers to a cell which requirescontact with a surface for growth or proliferation in vitro. Forexample, SAOS-2; U-20S; U-20S CycE; A172; T98G; U373MG; U87MG; SVGp12;BT-474; HMEC; MCF-7; MCF-10A; MDA-MB-231; MDA-MB-231M; MDA-MB-436;MDA-MB-468; SK-BR-3; T47D; ZR-75-1; MA11; PM1; SUM1315mo2; JIMT-1;HCC-1937; KPL-4; SUM-102PT; HeLa; HCT-116; SW480R18; SW480; HT29;Caco-2; SW620; DLD-1; LS174T; SW48; RKO; HCT-15; LS1034; AGS; CHO;SVpgC2a; HEK293; HEK293TREX; HA1ER; HA1EB; A549; A549EpoB40; U1690;A549EpoB480; NCI-H460; MDA-MB-435; UACC-257; NIH3T3; 1A9; 1A9/PTX10;1A9/PTX22; Ascites cells; KF28; KF28Tx; KFr13; KFr13Tx; Primary ovariansolid tumor cells; OVCAR-3; OVCAR-4; OVCAR-5; OVCAR-8; OVCAR-8/ADR;SU.86.86; CAPAN-1; Hs 766T; 22-RV-1; DuCaP; LAPC-4; LnCaP; Primaryprostate stromal cells; Primary prostate epithelial cells; MDA-P; CA-1;MDA-PCA-2b; PC-3; PC-3M; PWR-1E; RWPE-1; VCaP; WPE-1/NA22; WPE-1/NB11;WPM4-1; P97E; ALVA-31; RD; and A431. Generally, most cells derived fromsolid tissues are adherent cells (Rantala, J. K. et al. BMC Genomics12:162 doi:10.1186/1471-2164-12-162 (2011)).

A “suspension culture”, as used herein, refers to a culture in whichcells grow or multiply while suspended in a suitable fluid medium.Accordingly, a “suspension cell”, as used herein, refers to any cellwhich is cultured while suspended in a suitable medium. A person skilledin the art will understand that a suspension cell need not naturallyexist or multiply while suspended in a fluid medium, provided that thecell is adapted to grow or survive in suspension culture. Furthermore, aperson skilled in the art will understand that, while a suspension cellis generally non-adherent, a suspension cell may retain some ability toadhere to a surface while being cultured in suspension. Accordingly,adherent or weakly adherent cells may be cultured as suspension cellsunder appropriate conditions. Suspension cells may include, for example,Chinese Hamster Ovary (CHO) cells; K562; BAF3; HEK293; Sf21; Sf9; S2;primary bone marrow or bone marrow-derived cells; primary cord bloodcells, primary hematopoietic cells, primary hematopoietic stem cells;hybridoma cells or primary blood-born cancer cells. Suspension cells maybe hematopoietic in origin or may be adapted to suspension culture froman adherent cell line.

A “fluid injection channel”, as used herein, refers to any conduitthrough which fluid may be introduced into a chamber of the device. Afluid injection channel can be used to deliver any fluid to a chamberincluding cell suspensions, cell culture media, wash buffers, reactionmixes, factors, reagents, functionalized beads, etc.

An “auxiliary chamber”, as used herein, refers to any chamber subsidiaryto a cell capture chamber. Auxiliary chamber can be used for treatmentor assaying of a captured cell, or its isolated contents. Treatment caninclude cell preparation steps including culture, washing, lysis, andfractionation. Assaying may include DNA and RNA amplification anddetection, including mitochondrial PCR; genomic PCR; digital PCR,RT-PCR, RTq-PCR, multiple displacement amplification (DNA), rollingcircle amplification sequencing, degenerate PCR, molecular inversionprobes, molecular beacons, as well as other DNA/RNA amplification anddetection methods, in vitro transcription, ligation, immunochemistry;reporter expression analysis; hybridization studies; and so forth.Several auxiliary chambers may be connected, in tandem and/or inparallel, to a single cell capture chamber, such that multipletreatments may be performed on the contents of a single cell capturechamber. A valve may be positioned between an auxiliary chamber and thecell capture chamber, or between auxiliary chambers, to regulated fluidflow between chambers.

The ability to assay cell products that reside intracellularly orextracellularly is an important capability in cell biology. Recombinantmonoclonal antibodies (mAbs) are one example of a cell product and areused in biological assays (for example, cell characterization,diagnostic testing) and as therapeutics. Chinese Hamster Ovary (CHO)cells are widely used to produce mAbs (Jayapal, K. P. et al. ChemicalEngineering Progress 103, 40-47 (2007)), now favored in large part dueto the demonstrated clinical safety and efficacy of their proteinproducts.

An important bottleneck in the development of mAb production processesis the need to generate cell lines that produce large quantities ofantibodies. After transduction of the gene of interest, stable cloneselection with the desired product quality can take several months. Thisis normally the longest step in the development of a new proteinproduction process (Chartrain, M. & Chu, L., Current PharmaceuticalBiotechnology 9(6), 447-467 (2008)). Most production cell lines havebeen generated by performing limiting dilution of a transduced pool ofcells in multiwell plates (Chartrain, M. & Chu, L., CurrentPharmaceutical Biotechnology 9(6), 447-467 (2008); Browne, S. M. &Al-Rubeai, M., Trends in Biotechnology 25(9), 425-432 (2007); and RitaCosta, A. et al. European journal of pharmaceutics and biopharmaceutics74(2), 127-138 (2010)), with often >1,000 wells screened due to the lowcell plating efficiency (McCullough, K. C. et al. Journal of BiologicalStandardization 11(3), 183-194 (1983); and Porter, A. J. et al.Biotechnology Progress 26(5), 1455-1464 (2010)) and the need to analyzemany candidates. This method requires at least 2 weeks of culture toallow accumulation of detectable mAbs concentrations before a firstmeasurement can be made. Lowest producing clones are eliminated whilehighest producers are advanced to the next phase of scale-up, alaborious process that is often repeated for subsequent rounds ofsub-cloning to ensure the generation of clonal cell lines (Underwood, P.A. & Bean, P. A. Journal of Immunological Methods 107(1), 119-128(1988)). In an effort to increase throughput and accelerate theidentification of high-producing cells, several FACS-based methods havebeen developed. Cell sorting strategies can be coupled with single-celldeposition, hence eliminating the need for sub-cloning. These strategiesinclude immunolabeling of surface-mAb by immunostaining (Brezinsky, S.C. G. et al. Journal of Immunological Methods 277(1-2), 141-155 (2003))and the integration of a reporter gene into the vector (Mancia, F. etal. Structure 12(8), 1355-1360 (2004); Meng, Y. G. et al. Gene 242(1-2),201-207 (2000); Bailey, C. G. et al. Biotechnology and Bioengineering80(6), 670-676 (2002); and Pilbrough, W. et al. Plos One 4(12), 11(2009)), which can also be engineered to minimize the impact of thefluorescent protein on the translation of the desired product (Cairns,V. R. et al. Biotechnology and Bioengineering 108(11), 2611-2622(2011)). However, in some systems the transcription of reporters orsurface-bound mAb levels is poorly correlated with the amount ofsecreted mAb (Hanania, E. G. et al. Biotechnology and Bioengineering91(7), 872-876 (2005)). Therefore, methods have been developed todirectly measure the secreted proteins using gels or semi-solid mediumthat limit diffusion and maintain the secreted mAb molecules in thevicinity of the producing cells. For instance, single cells can beencapsulated in gel microdrops which are then subsequently labeled witha fluorescent antibody and sorted to select high producing cells(Powell, K. T. & Weaver, J. C. Bio/Technology 8(4), 333-337 (1990)). Asimilar approach is used with matrix-based secretion assays but theproduct is captured directly on biotinylated cells using an avidin-boundantibody (Manz, R., et al. PNAS 92(6), 1921-1925 (1995); and Borth, N.et al. Biotechnology and Bioengineering 71(4), 266-273 (2000).) and thenfluorescently labeled before sorting. Other secretion assays involvecultivating cells in semi-solid medium over multiple days, allowing theproduct to concentrate around single cells, resulting in a halo offluorescent-tagged antibody (Hanania, E. G. et al. Biotechnology andBioengineering 91(7), 872-876 (2005); Dharshanan, S. et al. ElectronicJournal of Biotechnology 14(2) (2011)). These methods require cells tobe seeded at low densities to ensure clonality in the semi-solid mediumsuch that these conditions may not select cells that will perform wellafter scale-up in suspension culture medium.

Miniaturization can accelerate secretion assays by rapidly concentratingthe products from single cells in small volumes while providing thethroughput needed for large screens. Microwell arrays have been reportedto screen for large numbers of antibody-secreting single cells (Love, J.C. et al. Nature Biotechnology 24(6), 703-707 (2006); Park, S. et al.Journal of Biotechnology 156(3), 197-202 (2011); Park, S. et al.Analytical Chemistry 82(13), 5830-5837 (2010); and Jin, A. et al. NatureMedicine 15(9), 1088-1092 (2009)). For instance, using microengravingcells are trapped into a microwell array and the secreted antibody iscaptured onto a functionalized glass cover that is then removed andstained prior to being scanned. This method has been multiplexed toassess levels of glycolysation in addition to secretion (Park, S. et al.Analytical Chemistry 82(13), 5830-5837 (2010)). However, selected cellsmust be cultured in an open array or transferred to multiwell plates forclonal expansion, both dilute conditions. Microfluidic devices capableof identifying single antibody-secreting cells isolated inpicoliter-volume aqueous droplets of chambers have been reported(Singhal, A. et al. Analytical Chemistry 82(20), 8671-8679 (2010); andKoester, S. et al. Lab on a Chip 8(7), 1110-1115 (2008)). However, anunderexploited feature of these enclosed systems is the use of smallvolumes to carry out clonal culture experiments at high seedingdensities. Cloning by inoculating one cell into 4 nl yields aconcentration of 250,000 cells ml⁻¹, thus a comparable seeding densityto conventional macroscale passaging. This high concentration canprovide a conditioning of the culture environment to potentially enhancethe cloning efficiency compared to limiting dilution cultures.Furthermore, the isolation of clones in nanoliter volumes usingintegrated microvalves (Thorsen, T. et al. Science 298(5593), 580-584(2002)) has the advantage of concentrating secreted proteins without theneed for a semi-solid matrix, thus allowing for rapid detection of mAbproduction. Most importantly, the immobilization of suspension cells bysequestering clones in high aspect ratio microfluidic chambers (Lecault,V. et al. Nature Methods 8(7), 581-586 (2011)) allows the cells to beassayed and cultured in liquid medium similar to bioreactor cultures.The operation of these microfluidic devices can easily be automated andcombined with time-lapse imaging to confirm clonality and trackproliferation. A microfluidic cell culture platform is shown to rapidlyassay the amount of secreted mAb from single cells and culture hundredsof clones simultaneously without the need for a semi-solid matrix. Theuse of this platform is shown to generate clonal cell lines from a poolof suspension-adapted CHO cells producing a recombinant IgG1 mAb.

Methods

The following methods were used for the fabrication of embodimentsdescribed herein in the examples disclosed below. It will be apparentthat other methods, materials and designs are possible for creatingother embodiments while remaining within the spirit of the inventiondescribed herein.

Microfluidic Cell Culture Array Fabrication

Devices were entirely made out of PDMS (Sylgard 1840, Dow Corning™). Thecell culture array, control, and membrane layers were assembled usingmultilayer soft lithography techniques (Unger, M. A. et al. Science 288,113-116 (2000); and Thorsen, T. et al. Science 298, 580-584 (2002).)while the iso-osmotic bath and cover layers were integrated by PDMSstamping (Satyanarayana, S. et al. J. Microelectromech. Syst. 1414,392-399 (2005)). Chips were covalently bound to glass slides by oxygenplasma treatment. Devices were left at 80° C. for at least 5 days andautoclaved prior to use for cell culture applications to drive thecuring reaction towards completion. Detailed protocols for mold anddevice fabrication are as follows.

Wafer Fabrication Protocol.

Each new microfluidic design is created with a drawing software such asAutoCAD. A micro-pump is located downstream of the array to avoidcrushing the cells and control the speed during the loading process.Depending on the application, microfluidic cell culture arrays maycontain from 1,600 to ˜20,000 chambers in the order of ˜4 nl each.Multiplexers, isolation valves, osmolarity regulator, hydration linesetc. can be added when necessary to offer a better control of themicroenvironment. Designs are printed at 20,000 dpi on transparentmasks. The fabrication of molds on a silicone substrate is performedusing common photolithography techniques as described below.

Flow Wafer

Flow Channels

-   -   1. Dehydrate a wafer for 10-15 minutes at 150° C.    -   2. Treat the wafer with vapor phase HMDS for at least 2 minutes.    -   3. Pour SPR220-7.0 resist on half the diameter of the wafer.    -   4. Ramp at 500 rpm for 10 seconds, then spin at 1,500 rpm for 90        seconds.    -   5. Pre-bake the wafer at 115° C. for 120 seconds.    -   6. Expose for 30 s.    -   7. Wait 30 minutes to rehydrate the resist.    -   8. Develop in MF319 primary bath for around 5-10 minutes, then        rinse in an MF319 secondary bath.    -   9. Rinse with DI water and dry the wafer with compressed        nitrogen.    -   10. Ramp from room temperature to 190° C. and leave overnight        for hard bake.    -   Aim: 11-13 μm after reflow

Inlet Channels

-   -   1. Pour SU8-50 resist on half the diameter of the wafer.    -   2. Ramp at 500 rpm for 30 seconds, then spin at 2,500 rpm for 30        seconds.    -   3. Soft bake the wafer for 2 minutes at 65° C., 10 minutes at        95° C., and 2 minutes at 65° C.    -   4. Expose for 7 s.    -   5. Perform a post-exposure bake for 2 minutes at 65° C., 10        minutes at 95° C., and 2 minutes at 65° C.    -   6. Develop in an SU8 developer primary bath for around 4        minutes, then rinse in a SU8 developer secondary bath.    -   7. Rinse with IPA and dry the wafer with compressed nitrogen.    -   Aim: 40 μm

Chambers

-   -   1. Pour SU8-100 resist on half the diameter of the wafer.    -   2. Ramp at 500 rpm for 10 seconds, then spin at 1,300 rpm for 50        seconds.    -   3. Soft bake the wafer for 5 minutes at 65° C., 70 minutes at        95° C., and 5 minutes at 65° C.    -   4. Expose for 25 s.    -   5. Perform a post-exposure bake for 5 minutes at 65° C., 18        minutes at 95° C., and 5 minutes at 65° C.    -   6. Develop in an SU8 developer primary bath for around 20        minutes, then rinse in a SU8 developer secondary bath.    -   7. Rinse with IPA and dry the wafer with compressed nitrogen.    -   8. Ramp up and down from room temperature to 135° C. for 20        minutes.    -   Aim: 160 μm

Control Wafer

-   -   1. Dehydrate a wafer for 10-15 minutes at 150° C.    -   2. Pour SU8-50 resist on half the diameter of the wafer.    -   3. Ramp at 500 rpm for 10 seconds, then spin at 4,200 rpm for 40        seconds.    -   4. Soft bake the wafer for 2 minutes at 65° C., 4 minutes at 95°        C., and 2 minutes at 65° C.    -   5. Expose for 2 minutes.    -   6. Perform a post-exposure bake for 2 minutes at 65° C., 6        minutes at 95° C., and 2 minutes at 65° C.    -   7. Develop in an SU8 developer primary bath for around 2        minutes, then rinse in a SU8 developer second bath.    -   8. Rinse with IPA and dry the wafer with compressed nitrogen.    -   9. Ramp up and down from room temperature to 135° C. for 20        minutes.    -   Aim: 25 μm

Device Fabrication Protocol

Cleaning

-   -   1. Place control wafers in plastic box with TMCS (can clean flow        wafers with PDMS, but that requires degassing) for at least 2        minutes.    -   2. Pour 15.0 g RTV-A and 1.5 g RTV-B (10:1 ratio) per wafer into        plastic cup, place cup in mixing machine, and mix together.    -   3. While machine mixing, wrap 1 Petri dish per wafer with        aluminum foil.    -   4. After mixing is done, remove wafers from TMCS box and place        in Petri dishes    -   5. Pour PDMS onto each wafer and tilt dish so that wafer is        covered with PDMS and that PDMS overflows on the foil.    -   6. Place in 80° C. oven for at least 20 minutes.    -   (Can be left overnight after performing this step.)

Flow Layer

-   -   7. Place flow wafers in plastic box with TMCS for at least 2        minutes.    -   8. Pour 12.5 g RTV-A and 2.5 g RTV-B per wafer in 5:1 plastic        cup, place cup in mixing machine, and mix together.    -   9. While machine mixing, prepare aluminum wrap using metal dish.    -   10. After mixing is done, remove wafers from TMCS box and place        in aluminum holders. Press down wafer on the bottom by folding        the aluminum foil on top of wafer edges.    -   11. Pour PDMS onto each wafer, and level the aluminum holder        with 2 micropipette tips.    -   12. Place into degasser machine, pressurize, and degas for until        no visible bubbles are left. Prepare control layer during that        time.    -   13. Remove from degasser and level again with 2 micropipette        tips. Let sit for at least 15 min.    -   14. Place in 80° C. oven for 18 minutes.

Control Layer

-   -   15. Cut around cleaned wafer with surgical knife and peel off        PDMS to release the cleaned wafer.    -   16. Place cleaned control wafer in plastic box with TMCS for at        least 2 minutes.    -   17. Pour 15.0 g RTV-A and 0.75 g RTV-B into 20:1 plastic cup,        place cup in mixing machine, and mix together.    -   18. Turn on gas and vacuum for spinner.    -   19. Ensure spinner recipe ramps in 5 seconds to 500 rpm, dwells        at 500 rpm for 10 seconds, ramps to 1630 rpm in 10 seconds,        dwells at 1630 rpm for 60 seconds, and ramps down to 0 rpm in 5        seconds.    -   20. Place wafer carefully on centre of spinner chuck, close lid        and secure with copper slab, and execute spinner recipe.    -   21. After spinning is finished, remove wafer from spinner and        place in clean, new Petri dish. Let sit for at least 15 minutes.    -   22. Place in 80° C. oven for 18 minutes    -   (The control and flow layers should go into the oven at the same        time.)

Membrane

-   -   23. Cut around cleaned wafer with surgical knife and peel off        PDMS.    -   24. Pour 15.0 g RTV-A and 0.75 g RTV-B into 20:1 plastic cup,        place cup in mixing machine, and mix together.    -   25. Turn on gas and vacuum for spinner.    -   26. Ensure spinner recipe ramps in 5 seconds to 500 rpm, dwells        at 500 rpm for 10 seconds, ramps to 500 rpm in 10 seconds,        dwells at 500 rpm for 60 seconds, and ramps down to 0 rpm in 5        seconds (A thinner membrane will result in leaky valves while a        too thick membrane does not spread evenly on the wafer).    -   27. Place wafer carefully on centre of spinner chuck, close lid        and secure with copper slab, and execute spinner recipe.    -   28. After spinning is finished, remove wafer from spinner and        place in clean, new Petri dish.    -   29. Let sit for at least 15 minutes and align flow/control        during that time.    -   30. Place in 80° C. oven for 12 minutes (13 min after        flow/control duo has been placed in the oven)

Flow/Control Alignment

-   -   31. Remove both flow and control wafers from the oven.    -   32. Cut inside the edge of the flow wafer with a surgical knife,        then peel off PDMS layer from silicon wafer.    -   33. Place control wafer under the microscope.    -   34. Align flow layer to control layer, trying not to peel off        and on too much.    -   35. Push down any bubbles that remain between the two layers,        and place in 80° C. oven for 25 min.    -   (The blank should come out of the oven at the same time as the        flow/control combo. Time out accordingly.)

Membrane/Duo Alignment

-   -   36. Remove both flow/control duo and blank wafers from the oven.    -   37. Cut around the edge of the control/flow wafer with a        surgical knife, then peel off PDMS layer from silicon wafer    -   38. Place flow/control duo onto blank layer.    -   39. Push down any bubbles that remain between the two layers,        and place in 80° C. oven for at least one hour.    -   (Can be left in the oven overnight after this step.)

Bath Layer

-   -   40. Pour 40.0 g RTV-A and 4.0 g RTV-B in 10:1 plastic cup, place        cup in mixing machine, and mix together (This amount of PDMS        gives a sufficient height to provide good support structure for        inlet and outlet ports.).    -   41. While machine mixing, prepare aluminum wrap using metal        dish.    -   42. Press down wafer on the bottom by folding the aluminum foil        on top of wafer edges.    -   43. Pour PDMS onto blank wafer, and level the aluminum holder        with 2 micropipette tips.    -   44. Place into degasser machine, pressurize, and degas until no        visible bubbles are left.    -   45. Remove from degasser and level again with 2 micropipette        tips.    -   46. Place in 80° C. oven for 20 minutes.

Cover Layer

-   -   47. Pour 14.0 g RTV-A and 1.4 g RTV-B in 10:1 plastic cup, place        cup in mixing machine, and mix together.    -   48. While machine mixing, prepare aluminum wrap using metal        dish.    -   49. Press down wafer on the bottom by folding the aluminum foil        on top of wafer edges.    -   50. Pour PDMS onto each wafer, and level the aluminum holder        with 2 micropipette tips.    -   51. Place into degasser machine, pressurize, and degas until no        visible bubbles are left.    -   52. Remove from degasser and level again with 2 micropipette        tips.    -   53. Place in 80° C. oven for 20 minutes.

Chip Assembly

-   -   54. Remove flow/control/membrane wafer, and blank wafers from        the oven and let cool for about 5 minutes.    -   55. Dice layers into individual chips and place the chips on a        ball bearing bed, flow layer down.    -   56. Dice the bath layer, and cut inside to create a bath having        the area of the array. Leave enough space for the ports and the        edges.    -   57. Punch holes that go in the corner of each side of the bath.    -   58. Dice the cover layers into pieces bigger that each chip.    -   59. Clean all surfaces with scotch-tape.    -   60. Mix together about 10.0 g RTV-A and 1.0 g RTV-B in 10:1        plastic cup, place in mixing machine, and mix together.    -   61. Set spinner to spin at 6,000 rpm for 6 minutes.    -   62. Remove blank wafer and place on spinner, pour on PDMS, and        spin.    -   63. Remove from spinner and place in Petri dish.    -   64. “Stamp” the bath portion onto the liquid blank wafer and        leave for 30 seconds. Make sure to stamp the right side of the        bath.    -   65. Remove from wafer, and stick together with the flow/control        portion.    -   66. Remove bubbles between layers.    -   67. Mix together about 10.0 g RTV-A and 1.0 g RTV-B in 10:1        plastic cup, place in mixing machine, and mix together.    -   68. Set spinner to spin at 6,000 rpm for 6 minutes.    -   69. Remove blank wafer and place on spinner, pour on PDMS, and        spin.    -   70. Remove from spinner and place in Petri dish.    -   71. “Stamp” the cover layer portion onto the liquid blank wafer        and leave for 30 seconds.    -   72. Remove from wafer, and stick on top of the bath portion.    -   73. Remove bubbles between layers    -   74. Leave chips to cure at room temperature overnight on ball        bearings and place them in the oven.    -   (After this step, the chips can be left in the oven.)

Hole Punching/Bonding to Glass

-   -   75. Remove chips from the oven and punch appropriate holes with        the hole puncher.    -   76. Clean glass slides with IPA and PDMS chips with Scotch tape.    -   77. Use plasma bonder to bond together chips and glass slides        (25 s).    -   78. Cure at 80° C. in oven overnight.

The total curing time at 80° C. should equal at least 5 days beforetesting of chips, and chips should be 12 days old and autoclaved beforeuse for cell culture.

Microfluidic Cell Culture

Microfluidic devices were placed inside a custom environmental chamber(Live Cell Instrument™, Chamide). The temperature was maintained at 37°C. with 5% CO₂ in humidified air. Humidity saturation was maintained bythe addition of two 3 cm-petri dishes filled with water inside themicroscope incubator. The iso-osmotic bath and the device were filledwith medium 24 hours prior to loading the cells to create equilibriumwith the environment. Positive pressure was maintained by gravity in theiso-osmotic bath by connecting a 3 mL syringe filled with medium to thebath, thus preventing the formation of air bubbles that could alterimaging (see FIG. 5). The content of the bath was replaced before cellloading but was not exchanged during the experiment. Assuming a relativehumidity of 90% in the microscope incubator, we calculated the waterlosses from the bath to be in the order of ˜1% over the course of a 5-dexperiment. Water vapor loss from the osmotic bath may be modeled as anear-Fickian diffusion and has a flux given by,

J=−D VC  (1)

where D is the diffusion constant of water vapor in PDMS (˜8.5×10⁻¹⁰ m²s⁻¹) and C is the concentration of water vapor in the bulk PDMS. Theiso-osmotic bath covers the area of the array (20 mm×11 mm) and has aheight of ˜5 mm. The majority of vapor loss occurs through the topsurface of the chamber that is sealed with a 1 mm thick layer of PDMSand through the long and short sides of the bath that are sealed with 5mm and 3 mm thick edges of PDMS respectively. This is well approximatedas a one-dimensional diffusion for problem given by,

J=−DΔC/L  (2)

where L is the thickness of the PDMS sealing the top and 4 sides of theosmotic bath. A saturated water vapor concentration at 37° C. on theinside surface of the membrane is assumed (˜39.3 mol m⁻³). Assuming a90% relative humidity in the incubator, the water vapor concentration atthe outside surface of the chip is approximated to be 0.9×mol m⁻³=35.4mol m⁻³, giving a total vapor flux of 2.1×10⁻⁸ g s⁻¹. This correspondsto a loss of 13 μl over a 5 day experiment. Given a total osmotic bathvolume of 1.1 ml this results in approximately 1.2% change in osmoticstrength during an experiment. Cells were concentrated to 2×10⁶cells/mL, transferred to a Teflon® tube and plugged in the device with astainless steel pin. The channels were flushed with medium and cellswere pumped into the device at a rate of 1 μL/min. Cells were allowed tosettle down in the chambers, then more cells were introduced until anadequate density was reached. In order to prevent air bubbles fromforming inside the device, an inlet pressure of 4 psi and an outletpressure of 1 psi were maintained at all times. When activated, pumpsand valves were pressurized at 35 psi.

For cultures of ND13 and NA10hd cells, filtered DMEM with 15% FBS, 1.6μg ml-1 puromycin, 100 ng ml-1 mouse SF, 10 ng ml-1 human IL-6 and 6 ngml-1 mouse IL-3 (all cytokines from STEMCELL Technologies™) wasexchanged by replacing four times the volume of the chip. Tests withfluorescent dye showed that this amount was sufficient to replace thevolume of the chip. Referring to FIG. 19, a microfluidic cell culturearray was loaded with medium supplemented with PE-TexasRed-streptavidinand the inlet was replaced by medium only. Pictures of the last 3columns of the array were taken during perfusion and fluorescenceintensity was quantified with Image J (National Institute ofHealth—Collins, T. J. BioTechniques 43 (1 Suppl): 25-30 (2007)). Thisdemonstrates that perfusing 4-fold the volume of the array (26 μl) issufficient for complete medium exchange. Each data point in FIG. 19represents the average of 9 wells and error bars represent the standarddeviation. Despite the low flow rates, the small length of the chambersallowed for efficient exchange of nutrients, growth factors andmetabolites through a combination of convection and diffusion. For smallmolecules (diffusion coefficient (D) was ˜10⁻⁹ m² s⁻¹) or proteins (Dwas ˜10⁻¹ m² s⁻¹), this diffusion time was approximated by τ of ˜x2/D,where x is one half the chamber height, giving exchange times of 10 s or100 s, respectively. These exchange times are substantially shorter thanthe 10-15 min periods used for medium perfusion.

Medium was exchanged by replacing 3 times the volume of the chip after24, 36, 48, 54, 60, 66 and 72 hours of culture. For single-cell culturesof ND13 cells, we found that medium exchanges at 24, 36, 48, 54, 60, 66and 72 h were sufficient to avoid conditions that led to decreasedgrowth rates (owing to nutrient limitations and/or build-up ofgrowth-inhibiting metabolites). Integrated micro-pumps and micro-valveswere automatically controlled by custom scripts (LabVIEW™, NationalInstruments). The average doubling time (τd) for each clone wascalculated by τd=72×ln(2)/ln(N72), where N72 is the number of cells perclone at 72 h. Primary E-SLAM cells were isolated as describedpreviously (Kent, D. G. et al. Blood 113:6342-6350 (2009)) and culturedin Iscove modified Dulbecco medium supplemented with 10 mg ml-1 bovineserum albumin, 10 μg ml⁻¹ insulin, 200 μg ml⁻¹ transferrin, 40 μg ml-1low-density lipoproteins, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹streptomycin, 2 mM glutamine (all from STEMCELL Technologies™), 10⁻⁴ Mβ-mercaptoethanol (Sigma™) plus 20 ng ml⁻¹ IL-11 (Genetics Institute)and SF, as indicated. Before starting the experiment, the volume neededto completely exchange the medium in the array (including the deadvolume from the medium inlets to the multiplexer) was tested using afluorescent dye. Ten minutes of perfusion was sufficient to remove thedye below detectable levels. Medium was exchanged every 2 h, and wepumped for 15 min for each condition to ensure that any medium remainingfrom a previous condition would be washed out of the array. Images weretaken every 12 min in two focal planes. Cell survival and early divisiontimes were assessed manually by looking at the videos, and theindividual growth curves for each clone were generated using the bifocalimage analysis algorithm described below. The content of the bath wasreplaced before cell loading but was not exchanged during theexperiment. Assuming a relative humidity of 90% in the microscopeincubator, the water losses from the bath were calculated to be in theorder of ˜1% over the course of a 5-day experiment.

Image Acquisition

The environmental chamber and the microfluidic devices were mounted ontoan inverted microscope (Axiovert 200™, Carl Zeiss™). Bright field imageswere acquired with a 20× objective and a CCD camera (Orca ER, Hamamatsu)connected to a computer. The entire microfluidic cell culture array wasautomatically scanned with a motorized staged (ProScan II™, PriorScientific) every 6 hours or selected wells were imaged every 5 min.

Alignment and Autofocus

Chamber alignment and autofocus scripts were implemented to acquirehomogeneous images, which in turn, improved the efficiency of cellsegmentation. Each of the 400 image frames contained 4 chambers. Thecoordinates of the 4 corners of the array were first determinedmanually; then coordinates for the entire grid were automaticallycalculated by extrapolation based on the device geometry. In order toadjust for small, local device distortions introduced during devicefabrication, each image frame was automatically aligned and focused. Foreach image frame, both a row and column average was calculated. The darkedges of the chambers produced reproducible valleys in these profiles.The locations of these valleys were then found and used to calculate theshift needed in order to align the wells to the image. Once cells wereloaded into the device, the images were automatically focused byminimizing the variance of the intensity of the pixels contained withineach chamber. A constant offset was then applied to each focus positionto increase the accuracy of the cell segmentation algorithm. Thesescripts were implemented in LabVIEW™ (National Instruments).

Image Analysis

To manage the large number of images generated per experiment, a customimage analysis software program was developed to automatically count thecells at each time point in individual chambers. Cell segmentationscripts were written in MATLAB™ (MathWorks™). Referring to FIG. 17 a,segmentation was accomplished through three main steps: chambersegmentation (A-E), cell-containing region segmentation (F-J), and thensingle cell isolation (K-O). First, the individual chambers aresegmented from the image background. This step of the segmentation isaccomplished by applying a bandpass filter (B) and then creating abinary image through an automatically determined threshold (C). Theresulting binary image is enhanced by removing objects touching theimage borders and suppressing noise by removing small objects (D).Finally, the chambers are segmented from the rest of the background byfilling in the holes created by the edges of the chambers. Next, theregions containing cells are separated from the rest of the chamber.This is achieved by first applying a local standard deviation filter toenhance the highly variable regions (G). The noise in the filterresponse is then suppressed by removing small regions, and this resultis converted into a binary image through an empirically determinedthreshold (H). Any holes in this result are then filled in to create thefinal region mask (I). To segment the individual cells from the rest ofthe group, a bandpass filter is applied to the output of a localstandard deviation filter applied to the image (K). A top hat filter isthen used to enhance the edges (L), and the bounded regions aresubsequently filled (M). This result is then converted to a binary imageusing an automatically determined threshold, and further enhanced byremoving small objects (N). FIG. 17 b shows the results of a comparisonbetween automated and manual cell counts, which demonstrated that theautomated cell count was in agreement with the manual quantification ofthe cells The straight line represents the 1:1 slope. Deviations athigher cell numbers are caused by the shadow around the edges somechambers, thereby resulting in a slight underestimate of cell numbersusing the image algorithm. An enhanced bifocal algorithm can correctthis error.

For experiments requiring a high count accuracy, for instance togenerate growth curves of primary HSCs, an enhanced cell-segmentationalgorithm was developed based on sets of images taken at two differentfocal points (˜50 μm apart). One image remained in focus, and the otherwas taken above the focal plane for use in segmentation. Aftersegmenting the well as described above, the portion of the image thatwas hidden by edge shadows was identified by comparing the intensity ofthe region inside the perimeter to the global mean intensity of thewell. The shadow was removed by calculating a brightness gradient maskaround the obstructed region, combining it with the well mask andapplying it to the original image. Next, the high-contrast image wasused to identify the center of cells, which appeared as high-intensityspots, by applying a brightness threshold. The centers were then dilatedto achieve accurate cell size representation. The focused image was usedto identify cell boundaries. The image intensity was inverted andsharpened using a negative Laplacian filter to enhance the cell edges.The sharpened image was then subtracted from the original, leaving onlythe cell contours and well. A bandpass size filter was then applied toremove objects that did not correspond to cell perimeters. The maskcontaining the cell contours was combined with the cell center mask, andthe image was dilated. A watershed cut algorithm was then applied toseparate adjacent cells that may have been connected during the dilationand filling processes Finally, the segmented image was compared to aninitial image without cells, and objects common to both were removed.This enhanced bifocal algorithm gave high-accuracy cell counts withexcellent correspondence to cell counts with excellent correspondence tocell counts determined by manual counting, as demonstrated in FIGS. 20 aand 20 b. FIG. 20 a shows a comparison between automated and manual cellcounts. The straight line corresponds to a linear least squareregression. FIG. 20 b shows absolute differences between the algorithmand manual counts.

Live Cell Immunostaining

For live cell immunostaining, the microincubator was turned off, and themain body containing the microfluidic device was placed on ice. For eachstep, at least ˜26 μL (4-fold the volume of the entire array) was pumpedinto the array to ensure complete replacement of the solution. Thedevice was filled with blocking solution for 20 min. The biotinylatedantibody cocktail (anti-B220, Gr-1, and Mac-1-biotin) was then pumpedinto the device followed by incubation of the device for 40 min, and wasthen flushed with a solution of Hank's Balanced Salt Solutionsupplemented with 2% fetal bovine serum (2% Hanks). APE-Texas-Red-streptavidin solution was then pumped into the device,which was then incubated for another 40 minutes, and flushed again with2% Hanks until all background fluorescence had disappeared. The arraywas then filled with fresh medium and placed on the microscope forimaging. Bright field and fluorescent images (exposure time, 1 second)were taken for the entire array.

Cell Recovery

Micropipettes were pulled from glass capillaries to a diameter rangingbetween 80 to 140 μm. At the end of an experiment, the cover layer wasdelaminated from the chip, and selected colonies were recovered bypiercing the membrane with a micropipette. To recover the entire contentof the microfluidic device, the chip was flipped upside down and flushedwith medium by pumping backwards at a rate of 1 μL/min. Cells were thenrecovered from the Teflon® tube and placed in a tissue culture plate forfurther analysis. To assess the efficiency of recovery, the plate wascentrifuged for 5 min at 400 g, the cells were allowed to settle for 1hour and then manually counted using an inverted microscope.

Macroscale Cultures

ND13 cells (Pineault, N. et al. Leukemia 19, 636-643 (2005)) werecultured in the same medium as in the microfluidic device (e.g. DMEMwith 15% fetal bovine serum supplemented with growth factors (100 ng/mLmurine stem cell factor, 10 ng/mL human interleukin-6, 6 ng/mL murineinterleukin-3 and selected by puromycin). Cells were passaged every 2-3days and kept in culture for at most 60 days post-infection. Controlgrowth curves were generated with the help of an automated cell cultureanalyzer (Cedex™, Innovatis™). For single cell control cultures, cellswere diluted to a concentration of 5 cells/mL, and separated in 200 μLcultures in a U-shaped 96-well plate. Cells were centrifuged at 400 gfor 5 minutes and allowed to settle for one hour in the incubator. Wellscontaining single cells to start with were counted manually every 12hours. For colony-forming cell assays, approximately 720 cells(corresponding to 11 starting cell equivalents) were recovered from themicrofluidic array or conventional 96-well plates after 72 h in cultureand plated into triplicate methylcellulose assays for 14 d (MethoCult3484™, STEMCELL Technologies™), after which the number of coloniesobtained was manually counted under a microscope.

In Vivo Hematopoietic Reconstitution Assays.

Bone marrow cells obtained from C57Bl/6Ly-Pep3b mice were highlyenriched (−50% purity) for HSCs (Kent, D. G. et al. Blood 113, 6342-6350(2009)), and a total of 50 cells (representing 25 HSCs) wereretrovirally transduced with a NUP98-HOXA10hd retroviral vector andcultured for 11 days as previously described in Ohta, H. et al.(Experimental Hematology 35, 817-830 (2007)). On day 11, the cells wereharvested and split equally between cultures in a 96 well dish (control)or a microfluidic array for a further 3 days of culture. Cells wereharvested from both conditions, and then fractions representing1/1,520^(th) or 1/15,200^(th) of the starting cells (estimated as alimiting dose of HSCs assuming a minimum of 60-fold or 600-foldexpansion during the culture period respectively) were transplanted intolethally irradiated (810 cGy of x-rays) C57Bl/6-C2J mice along with100,000 BM helper cells. Six weeks, and 3 and 5 months later, peripheralblood samples obtained from each recipient were analyzed for evidence ofdonor-derived (GFP⁺) lymphoid and/or myeloid cells as follows.Erythrocytes were lysed with ammonium chloride (STEMCELL™) andleukocytes were suspended in 2% Hanks (STEMCELL™) and then incubatedwith a combination of PE-labeled anti-Ly6G/Mac-1, perCP-Cy5.5-labelledanti-B220 and APC-labeled anti-CD4/CD8 (BD Pharmingen™). Flow cytometricanalysis was then performed on a FACSAria (Becton-Dickinson™)

Transport Equations for Mathematical Modeling.

The simulation of the system was performed with a three-dimensional,steady state, single phase, laminar flow model. The CFD (computationalfluid dynamics) simulation has been done using FLUENT™ 6.3.26 (FluentInc.™). In laminar flow the Navier-Stokes equations describe themomentum transport. Therefore, the conservation of momentum in themicro-bioreactor is described by Eq. (3)

$\begin{matrix}{{{\frac{\partial}{\partial t}\left( {\rho\forall} \right)} + {x \cdot \left( {\rho {\forall\forall}} \right)}} = {{- {\nabla P}} + {\nabla{\cdot \overset{\overset{\_}{\_}}{\tau}}}}} & (3)\end{matrix}$

The conservation of mass is described by the continuity equation asfollows,

$\begin{matrix}{{\frac{\partial\rho}{\partial t} + {\nabla{\cdot \left( {\rho \; \overset{\rightharpoonup}{V}} \right)}}} = 0} & (4)\end{matrix}$

where ρ (Kg m−3) is the fluid density, V (m s-1) is the velocity vectorof the fluid, P (Pa) is the pressure, and τ is the stress tensor. Waterhas been used as a model to estimate the physical properties of fluid at37° C.

Boundary Conditions for Mathematical Modeling

The uniform velocity profile has been defined as the inlet boundarycondition. At the outflow boundary, the diffusion fluxes for all flowvariables in the direction normal to the exit plane are assumed to bezero. The fluid temperature is assumed to be constant at 37° C., and ano-slip boundary condition has been specified for the velocity at thewalls.

Statistical Analysis

Error bars were calculated using s.d. of the mean. Relative risk and 95%confidence intervals for the Cox proportional hazard model werecalculated using the ‘coxph’ function from the R package ‘survival’ withtied times of death being handled using the Efron approximation.

Maintenance Culture and Medium

A pool of CHO—S cells producing a human monoclonal IgG1 antibody Cellswas maintained in shake flasks with growth medium constituting of CDOptiCHO (Gibco, Life Technologies) supplemented with 15% CHO CDEfficientFeed A (Gibco, Life Technologies), 15% CHO CD EfficientFeed B(Gibco, Life Technologies), 4.5 mM L-glutamine (EmbryoMax, Millipore),15 μg ml⁻¹ puromycin (Sigma-Aldrich), 0.1 mM hypoxantin and 16 μMthymidine (Gibco, Life Technologies). Shake flasks were maintained at37° C. in a shaking incubator at 125 rpm (Minitron, Infors) with 6 or10% CO₂. Cells were passaged every 2-3 days and seeded at aconcentration ranging from 2-5×10⁵ cells ml⁻¹.

Growth Controls in Cloning Medium

For batch growth controls, cells were seeded at a concentration of2.5×10⁵ cells ml⁻¹ and cultured in 20 ml of cloning media consisting ofRPMI medium (Gibco, Life Technologies) supplemented with 17% CD OptiCHO(Gibco, Life Technologies), 3.75% CHO CD EfficientFeed A (Gibco, LifeTechnologies), 3.75% CHO CD EfficientFeed B (Gibco, Life Technologies),1 mM L-glutamine (EmbryoMax, Millipore), 10 μg ml⁻¹ insulin EMDMillipore, 5 μg ml⁻¹ transferrin (CellPrime rTransferrin AF, Millipore),2 g 1⁻¹ albumin (Cell Prime rAlbumin AF-G, Millipore) and 15 μg ml⁻¹puromycin (Sigma-Aldrich). Samples were taken daily and the viable cellconcentration was measured by an automated cell counter (Cedex,Illumina). To measure growth rates from single cells, a U-shaped nontissue culture-treated 96 well-plate (BD Falcon) was rinsed with cloningmedium. Cells were diluted to a concentration of 5 cells ml⁻¹ and 200 μlwas deposited in each well, equivalent to 1 cell per well. Cells werecentrifuged for 5 min at 400 g and left in the incubator for 1-2 h.Wells were manually scored to identify the starting number of cells ineach well. The number of cells in wells initially containing a singlecell was counted manually every day.

Microfluidic Cell Culture

Microfluidic cell culture arrays containing 1,600 chambers of 4.1 nl(160 μm×160 μm×160 μm) were fabricated as described in Appendix B. Twodays prior to loading the cells, the iso-osmotic bath was filled withcloning medium supplemented with penicillin (100 U ml⁻¹) andstreptomycin (100 μg ml⁻¹). The device was connected, primed withcloning medium and maintained in an environmental chamber (Chamide,LiveCell Instruments) at 37° C. and with 5% humidified CO₂. Two petridishes filled with water were added in the environmental chamber tomaintain the humidity. Cells were centrifuged for 5 min at 167 g andresuspended at a concentration of 2×10⁶ cells ml⁻¹ in fresh cloningmedium. Cells were then loaded in the microfluidic cell culture array byan approximately 1 μl min⁻¹ flow using an integrated micro-pump. Oncethe array was filled, cells were allowed to settle by gravity to thebottom of the chambers for ˜3 min. If needed, this process was repeateduntil a desired number of single cells was obtained. After the beadimmunocapture assay, the cells were cultured in a batch mode with theisolation valve kept open. Microvalves and image acquisition werecontrolled by custom scripts available upon request (LabVIEW, NationalInstruments).

Bead Immunocapture Assay

Cloning medium supplemented with 100 U ml⁻¹ penicillin and 100 μg ml⁻¹streptomycin was used throughout the bead immunocapture assay.Polystyrene protein-A coated beads with an average diameter of 4.9 μm(ProActive® Microspheres, Bangs Laboratories) were washed at least 4times with cloning medium by successive rounds of centrifugation at 7431g. The beads were resuspended at a concentration of 0.1 mg beads ml⁻¹and immediately loaded in the device at a flow rate of 2 μl min⁻¹ from apolytetrafluoroethylene tubing maintained in an upright position. Oncethe array was filled, the flow was stopped and the beads were allowed tosettle by gravity to the bottom of the chambers, resulting in an averageof 100-150 beads per chambers. Excess beads in the channels were flushedwith cloning medium for 5 min at a flow rate of 2 μl min⁻¹. Theisolation valves were then closed to sequester each chamber and thebeads were incubated with cells for 2 h at 37° C. The array was thenflushed with cloning medium for 15 min at a flow rate of 2 μl min⁻¹. Asolution of 20 μml⁻¹ labeled antibody (Dylight 594-conjugated AffiniPureF(ab′)₂ fragment of rabbit anti-human IgG (H+L) (Jackson Immunoresearch)diluted in cloning medium was first desalted by centrifugation (AmiconUltra 0.5-ml 100K, Millipore) and then pumped into the device for 15 minat 2 μl min⁻¹. The fluorescent antibody was incubated for 15 min, andthen washed with cloning medium for 30 min at 2 μl min⁻¹ before imaging.

Automated Image Acquisition

Custom scripts (LabVIEW, National Instruments), were developed to allowautomated image acquisition of the array divided into 400 framescontaining 4 chambers each. The location of every frame was determinedby selecting the four array corners, and then extrapolating thecoordinates of each frame. An alignment script (Lecault, V. et al.Nature Methods 8(7), 581-586 (2011)) was used to correct for the minordistortions in the devices that can be introduced during fabrication soas to position the chambers correctly within each frame. After loadingthe beads, the focus on each frame was automatically determined bytaking a stack of images and identifying the focal point providing themaximum pixel standard deviation within the chambers. Following the beadimmunocapture assay, a bright field image and a red fluorescent image(10 ms exposure) were taken for every frame. Frames were subsequentlyimaged in bright field every 30 min during cell culture.

Image Analysis Algorithms

A graphical user interface (GUI), was developed in Matlab (MathWorks) toquantify the CHO cell protein production by measuring the fluorescenceintensity emitted from the beads. Since each frame contained 4 chambers,the well boundaries were first identified by blurring the bright fieldimage and subtracting it from the original. This process enhanced areasof high spatial frequency such as the well edges. The wells were thenfilled and any object smaller than 1250 pixels were eliminated using asize filter. Artifacts caused by the presence of beads were corrected bydilation and the well edges were adjusted by erosion before creating amask image of the wells. A bead mask was also created from the brightfield image. The beads appeared to be much darker than the background,which allowed their segmentation using a brightness threshold. Allpixels with intensity below the threshold were set to one, indicatingthe pixel was part of a bead, while those above the threshold were setto zero. Often, the center of the beads appeared as bright spots in themask image due to diffraction, causing the beads to look like rings.These rings were closed and filled by a dilation step followed byerosion. Finally, any lone pixels, i.e. a single pixel with a value ofone surrounded by pixels with values of zeros, were removed from themask. A series of 6 fluorescent images containing empty wells were takenfor flat-field correction. The pixel intensity at each location wasaveraged for the set of images and used to correct for backgroundfluorescence. Next, the bead and well masks were combined and applied tothe fluorescent image by multiplying each pixel in the mask image withthe corresponding pixel in the fluorescent image. The total fluorescenceintensity was calculated by summing the values of all the pixels in theresulting image. The GUI enabled the user to correct for segmentationerrors when needed. The GUI also allowed the user to make a cell mask bytracing the contours of each cell. This mask was then applied to thefluorescent image to measure the intensity of each cell. The totalintensity of the both the beads and each cell, along with the samevalues normalized by the corresponding mask area, were saved foranalysis. The mean intensity of each well was calculated as the totalintensity divided by the total bead area. For the selection ofantibodies, stained beads were deposited on glass slides under acoverslip and imaged. The same algorithm was used to generate the beadmask from bright field images and to measure the fluorescence of thebeads (no chamber mask was applicable).

Clone Recovery and Expansion

Micropipettes with a tip diameter ranging from 50-100 μm were made fromglass capillaries. Prior to cell recovery, the cover layer of the chipwas cut inside the area of the bath and removed. Selected clones wererecovered with the micropipette using an oil microinjector (IM-9B,Narishige) and deposited in a U-shaped non tissue-culture-treated96-well plate containing 200 μl of cloning medium supplemented with 100U ml⁻¹ of penicillin and 100 mg ml⁻¹ of streptomycin in each well toprevent contamination from the recovery process. Clones were centrifugedfor 5 min at 400 g and incubated for 9 days. Viable clones were thentransferred to a 24-well plate containing 1 ml of growth medium. After 5days of culture, the plate was centrifuged for 5 min at 400 g and thesupernatant was recovered for titer analysis. The clones were thentransferred to 6 ml of culture medium in 6-well plates. Once confluent,the selected clones were expanded in a 20 ml shake flask culture andbanked as described above.

Batch Shake Flask Cultures

The selected clones were thawed rapidly and resuspended in 20 ml ofgrowth medium in shake flasks. The cells were cultured and passageduntil the viability reached more than 95%. The day before starting shakeflask studies, the cells were seeded at a concentration of 5×10⁵ cellsml⁻¹. The next day, the cells were seeded at 5×10⁵ cells ml⁻¹ in 20 mlof growth medium in duplicate flasks. Cell concentration was measuredwith an automated cell counter (Cedex, Illumina) on days 5 and 7 forclones generated by limiting dilution and days 3, 5 and 7 for clonesgenerated from the microfluidic platform. As well, a 1 ml-sample wastaken from each flask, centrifuged at 7,341 g and the mAb concentrationin the supernatant titer was measured as described below. The integralviable cell density (IVC) was calculated as follow:

IVC_(i+1)=0.5×(C _(i+1) +C _(i))×(t _(i+1) −t _(i))+IVC_(i)  (4.1)

where C is viable cell concentration (cell ml⁻¹) and t is the time inculture (days). The titer was plotted against the IVC and the slope upto day 5 was used to calculate the cell specific productivity (SPR).

Measurement of mAb Titers

Secreted mAb titers were measured by bio-layer interferometry using theOctet RED96 Analysis System (Forté Bio, Pall Life Sciences) with ProteinA Biosensors (Forté Bio, Pall Life Sciences). A standard curve a frompurified mAb standard of known concentration was generated with eachrun, ranging from 0-50 μg for low concentration samples and 0-500 μgml⁻¹ for high concentration samples. Supernatants exceeding the dynamicrange were diluted appropriately and reanalyzed.

Statistical Analysis

The error bars were calculated using the standard deviation of the mean.A two-tailed unpaired t-test with unequal variance calculated thecloning efficiency P values. The coefficients of determination (R²) werebased on least-square linear regressions. The coefficient of variation(CV) was calculated as the standard deviation over the mean. Thetheoretical Poisson distribution was calculated as follow:

$\begin{matrix}{{P(x)} = \frac{^{- \mu}\mu_{c}^{x}}{x!}} & (4.2)\end{matrix}$

where P(x)=probability of having a chambers containing x cells

-   -   x=number of cells per chamber    -   μ_(c)=average number of cells per chamber        The antibody binding curve to the bead was calculated using the        following Langmuir equation:

$\begin{matrix}{I = {I_{\max}\left( \frac{K \cdot c_{Ab}}{1 + {K \cdot c_{Ab}}} \right)}} & (4.3)\end{matrix}$

The constants I_(max) and K were determined by a Langmuir regressionusing the following equation:

$\begin{matrix}{\frac{c_{Ab}}{I} = {\frac{c_{Ab}}{I_{\max}} + \frac{1}{K \cdot I_{\max}}}} & (4.4)\end{matrix}$

where I=Bead fluorescence intensity

-   -   I_(max)=Maximum bead fluorescence intensity    -   K=Equilibrium constant (ml μg⁻¹)    -   c_(Ab)=IgG1 antibody concentration (μg ml⁻¹)

EXAMPLES Example 1.1 Design of a Microfluidic Device for Suspension CellCulture

Referring to FIG. 1, a schematic drawing of a microfluidic deviceaccording to one embodiment is shown generally at 10, with micrographsas insets. The microfluidice device 10 comprises an array of 1,600chambers 12, each having a volume of 4.1 mL with integrated microvalvesto allow precise control and exchange of media. The chambers 12 areconnected by flow channels 14. Hydration lines 16 are located on eachside of the array to minimize edge effects. Control lines consist of anisolation valve 18 and control lines (for example, a peristaltic pump)20 to control cell loading and perfusion rates. Fluid can be introducedto the microfluidic device 10 through an array inlet 22 in order toaccess the flow channels 14 and chambers 12. Fluid may leave the devicethrough an array outlet 24. Arrows point at single cells. The left scalebar represents 1 mm and the right scale bar represents 100 μm.Alternative embodiments could contain 1 to 50,000 chambers with volumesranging from 1 mL to 20 μL. Alternatively, if one large chamber wasconnected by flow channels on top the volume may be about 5 mL. Chambergeometries exploit the properties of laminar flow to allow forimmobilization of non-adherent cells without significant mechanicalstress during and between medium exchanges. Various embodiments of thedevice also allow facile and efficient recovery of the pooled orindividual contents of the chambers.

In order to exploit microfabrication methods that allow denseintegration of microvalves (Unger, M. A. et al. Science 288, 113-116(2000); Duffy, D. C. et al. Analytical Chemistry 70, 4974-4984 (1998);Thorsen, T. et al. Science 298, 580-584, doi:10.1126/science.1076996(2002)), PDMS was chosen as a preferred material. Other biocompatiblepolymers such as poly(methyl methacrylate) (PMMA),poly(L-lactic-coglycolic acid) (PLGA) or poly(glycerol sebacate) (PGS)PDMS could also be used for fabricating similar devices. In addition itwill be appreciated by one skilled in the art that other materials suchas alternative elastomers, polymers, semiconductors, or glass could beused. FIG. 2 is a schematic diagram of the layers that are assembledduring the fabrication of microfluidic device. The previously mentionedproblems of microfluidic devices made from PDMS were address byincorporating an integrated iso-osmotic bath 32 into the design ofmicrofluidic device. This was achieved by fabricating the nanovolumechambers in the chamber layer (cell culture array) 38 and controlstructures in the control layer 36 under a 150 μm thick PDMS membrane 34that separates them from an “iso-osmotic bath” 32 consisting of amacroscopic chamber filled with medium (˜750 μL in volume) and enclosedby a gas-permeable PDMS cover layer 30 to keep the bath sterile. Thecell culture layer, control layer and membrane were bound to each otherby multilayer soft lithography while the membrane, iso-osmotic reservoir32 and cover layer 30 were assembled through PDMS stamping. The PDMSchips were then bound to a glass slide 40. The integrated iso-osmoticbath 32 reservoir was filled with medium to prevent evaporation andmaintain constant osmolarity inside the chambers. The iso-osmotic bathcan be filled with medium and pressurized by gravity to avoid formationof air bubbles. The bath can, in some examples, be scaled proportionallyto fit the area of the cell culture array, and the membrane can rangefrom less than 1 μm to 5 mm thick depending on the application and thechoice of material. The relatively high volume ratio of the osmotic bathto the culture volume (˜100 times) and the lower surface to volume ratioof the osmotic bath as well as the near-saturation humidity provided bythe microscope incubator, together allow a preferred osmotic strength tobe maintained in each microculture for many days. Continuous exchangethrough the membrane also keeps PDMS-permeable medium components inequilibrium and dilutes any potentially toxic organic molecules into thelarge volume of the osmotic bath (Regehr, K. J. et al. Lab on a Chip 9,2132-2139, doi:10.1039/b903043c (2009)). In a an embodiment a staticbath may be used. However, smaller bath volumes could also be used ifthe bath content was exchanged frequently. This could for instance bedone using channels overlaying the chambers that are refreshed with newmedium. In an embodiment illustrated in FIG. 5, the bath content can bereplaced by removing a bath plug 13 and introducing fresh medium from apump, such as syringe 19, which may connect to the array via bath inlet17. In this embodiment, the array inlet 22 and array outlet 24 arepressurized by air and control lines 20 are connected to solenoidactuators and rest on a glass plate 40.

Example 1.2 Cell Immobilization

Various embodiments allow for perfusion of cells without disturbing cellposition. This capability may be exploited for experiments requiringdynamic medium exchange or immunolabeling of the cells during or at theend of an experiment. This is particularly useful for suspension cells.

Referring to FIG. 3, one embodiment shows a microfluidic device is anon-perturbing microfluidic cell capture and retention mechanism thatuses gravity to trap cells 50 in chambers with an inverted geometry withflow channels 14 running over the top and control lines 18. In thedisclosed embodiment, the chambers 12 have cubic dimensions of 160μm×160 μm×160 μm. However, larger (up to 1 mm×1 mm×1 mm) or smaller(down to 10 μm×10 μm×10 μm) could be used depending on the cell type andthe intended application of the device. The chambers according to thisembodiment have an aspect ratio of 1:1. However, chambers having anaspect ratio as low as 0.5 may be utilized to minimize shear forces onthe cells (for example, when non-adherent cells are used).

The chamber dimensions and flow rates may be designed to ensure that apermissible maximum force (for example, shear force) is exerted on cellsduring medium exchange and this may be adjusted as appropriate dependingon the cell types being used. If cells are completely non-adhering thechambers may be designed such that the forces do not produce anysignificant motion. The degree of motion considered significant will bedictated by the application for which the device is being used. Forexample, in an imaging application or manual cell lineage analysis itmay be required that the cells move less than one diameter between imagecapture events which may be anywhere from seconds to several hours todays. The microfluidic cell culture arrays may exploit laminar flow todeliver the cells to the chambers and then to ensure that the cells arenot disturbed by subsequent perfusion. During medium refreshment or cellloading the volume expansion from the flow channels (≦13 μm×100 μm) tothe chambers (160 μm×160 μm) creates a large reduction in velocity thatdrops off quickly to very low levels at the bottom of the culturechambers (e.g. FIGS. 3 and 4). In alternative embodiments where thedepth of the chamber (i.e. length of the shortest distance between theretaining position and first region) was 80 μm (y) and the length of thefirst region was 160 μm (x), it was observed that the velocity of theperfusion fluid at the retaining position was such that the cells at theretaining position were being moved by the flow of perfusion fluid. Theflow channel could be positioned at different heights within thechamber, as long as the bottom of the chamber is far enough from thechannel so that the velocity at the bottom of the well is low enough tomaintain cells immobilized by gravity. In the tested embodiment, thesuspended cells were first loaded into the array using themicrofabricated peristaltic pump 20 (FIG. 1) at an overall flow rate of1 μL/min. This corresponds to a maximum velocity of ˜1 mm/sec and shearstresses of <0.3 Pa (not shown), which is well below levels that elicitphysiological responses (Ma, N. N. et al. Biotechnology andBioengineering 80, 428-437, doi:10.1002/bit.10387 (2002)). Syringepumps, manual, gravity or pneumatic pressurization could be used asalternatives to the integrated micropump to control the flow rates. Bothpulsatile and continuous flows may be used to ensure minimal cell motionduring medium exchange. During loading, cells essentially follow thestreamlines at the top of the chambers, and thus pass through the arraywithout having the time to settle in the chambers. Once the array isfilled, the flow is stopped, and this then allows the cells to settleinto the bottom of the chambers where they are sequestered from the flowstreamlines. When necessary, cells may be concentrated on the chip byrepeating this loading process in a step-wise fashion. Typical loadingefficiencies of 10-30% of chambers may be achieved for clonal analyses(i.e., approximately 160-480 single cells per device). A person of skillwould be able to direct the desired number of cells to a chamber byadjusting cell concentrations, flow rates, flow times, etc. Celltrapping cups could be integrated in the flow channels to increase theseeding efficiency in other embodiments. Additionally, other trappingmechanisms, including dielectric forces, magnetic forces, and opticalforces could be used as appropriate. Alternatively, valve structurescould be designed to deterministically place cells in chambers.

In the preferred embodiment, medium exchange through the array at a flowrate of 2 μL/min, results in a maximum shear stress <10⁻⁴ Pa at adistance of one cell diameter from the chamber bottom (not shown).Direct observation of cells in arrays being perfused at this rate toexchange media or for immunolabeling showed that the positions of thecells remained undisturbed (FIG. 9 a), thereby validating the use ofthese strategies while monitoring the growth of individual clones. Thiscapability is demonstrated in FIG. 9 b where frequent imaging (<5 min)was used to track the progeny of three single HSCs and build celllineage trees over 60 hours while replacing the cell culture media every6 hours (see FIG. 9 c). The frequency of image acquisition can beadjusted based on the number of wells being observed and the timerequired to capture images of all the chambers.

In an embodiment described herein, the small chamber length-scale allowsfor efficient exchange of nutrients, growth factors and metabolites by acombination of convection and diffusion. For small molecules (D˜10⁻⁹m²/sec) or proteins (D˜10⁻¹⁰ m²/sec), this diffusion time isapproximated by τ×x²/D where x is one half the chamber height, givingexchange times of 10 sec or 100 sec, respectively. This is significantlyshorter than our medium perfusion protocols that have refresh times of10 minutes.

Recovery of Cells Post-Culture

Cell recovery is often required to enable functional assays to beperformed on the progeny of the input cells, or to select cells ofinterest for larger scale culture. A method to recover defined clonalpopulations is therefore a critical requirement for many applications ofmicrofluidic cultures.

FIG. 4 shows a numerical simulation of the flow profile through aculture chamber of an embodiment. With a flow rate of 0.0625 μl/minthrough the flow channel, the velocity in mm s⁻¹ for the flow channel 14is 2.4 mm s⁻¹ at the center of the flow channel, with a gradual decreaseat the edges of the flow channel to about 1.0-1.2 mm s⁻¹. The suddenexpansion when the fluid moves from the flow channel to the chambercreates a velocity drop, and the velocity in the cell retaining regionis reduced to less than 50 μm/s. The velocity in mm s⁻¹ for the culturechamber 12 ranges from about 1.6 to about 0.4 mm s⁻¹ immediatelyadjacent the inlet and outlet (see bright flares) of the flow channeland the remainder of the culture chamber 12 ranges from about 0.4 toabout 0.0 mm s⁻¹. The culture chamber 12 dimensions (160 μm×160 μm),flow channel 14 dimensions (100 μm×13 μm) and culture chamber 12 volume(4.1 nl) are also shown. The modeling predicts minimal flow rates at thebottom ⅚ of the chamber. In accordance with embodiments, thegravitational forces on the cells is greater than hydrodynamic forcesand cells remain in the cell retaining region while the perfusion fluidexits the chamber through the flow channel outlet. Similarly, modelingof fluid velocity during cell loading (modeled for a total flow rate of1 μL/min) suggests that a maximum velocity in the flow channels 14 doesnot exceed 1.2×10⁻³ m/s and that the maximum velocity in the majority ofthe chamber 12 is at or near 0 m/s (not shown) during cell loading. Whenthe flow is stopped, cells settle down in the chamber 12 by gravity tothe cell retaining region. Additionally, modeling of the sheer stress(Pa) on the channel walls 14 suggests during cell loading, the flow rateof 0.03 μl/min through the flow channel results in a maximum shearstress exerted on the cells is 0.3 Pa next to the channel wall (notshown). Similarly, modeling of sheer stress on cells within a chamber 12during media exchange (i.e. perfusion) at a flow rate of 0.0625 μl/minthrough the flow channel suggests that the maximum shear exerted on thecells while at the bottom of the chamber (i.e. cell retaining region)during medium exchange (based on a total flow rate of 2 μl min⁻¹) doesnot exceed 3.1×10⁻⁴ Pa and would be about 0.0004 Pa in the middle of thecell retaining region.

Embodiments of the chamber design and microfluidic apparatus describedalso allow for facile recovery of cells from the entire array by simplyinverting the device, causing the cells to settle into the higher-flowrate regions of the chambers (as shown in FIG. 4) and then recoveringthe pooled population by flushing back through the input port. Thisrecovery method is simple and efficient, allowing for the harvesting ofapproximately 90% of cells with losses mainly attributable to thenonspecific adherence of cells on the surface of chambers. However, whenselective recovery of the contents of specific individual wells isdesired, the layer of PDMS covering the osmotic bath can first beremoved and a sterile micropipette then used to pierce the membrane overany selected chamber followed by aspiration of its contents (not shown).This method was found to be remarkably reliable and easy, allowing morethan 90% of the cells in each well harvested to be recovered asdetermined by direct cell counts before and after (FIGS. 9 a and 9 b).It can be performed either manually of automatically if greaterthroughput is needed. Furthermore, aspiration through a capillary(controlled with micron precision) or optical forces could be used torecover select cells. Alternatively, cells could be labeled individuallyusing optical means while in the device, recovered together by flowingout of the device, and then subsequently identified using the marker.One such marker may be a fluorophore that changes spectral propertieswhen illuminated by a light source such as a focused laser or otherlight source capable of selectively labeling cells.

Example 1.3 Culture of Hematopoietic Cells

Culture of Single Hematopoietic Cells in Microfluidic Cell CultureArrays.

We tested the applicability of this microfluidic device to the studyprimitive hematopoietic cells. We first examined the growth of apreleukemic murine cells created by genetically engineering primitiveadult mouse bone marrow cells to express a NUP98-HOXD13 (ND13) fusiongene^(9,10). Matched cultures of these “ND13” cells were set up in24-well plates seeded at 150,000 cells/mL, 96-well plates seeded withsingle cells, and microfluidic cell culture arrays with or without theintegrated iso-osmotic bath. In the presence of the iso-osmotic bath,the population doubling time averaged over all chambers loaded withsingle cells faithfully reproduced the bulk growth rate seen in theculture plates, indicating comparable conditions had been achieved. Inaddition, the average rates of expansion of the clones generated in themicrofluidic chambers were equivalent to the average growth ratesobtained in the 200 μl 96-well cultures (FIG. 10). However, in devicesthat lacked the iso-osmotic bath, cell division and survival wasseverely compromised (e.g. FIG. 10), in spite of humidity control in themicroscope incubator and the initiation of medium exchanges 24 hoursafter starting the experiment, indicating that permeation effects occurwithin hours, which can in turn affect cell growth. In cases where othermaterials are used that have reduced transport properties the osmoticbath may not be required. Alternatively, the use of perfusion withsufficient frequency may be used to reduce the need for the osmoticbath.

A single cell in a 4.1 nL isolated chamber is at an effectiveconcentration ˜2.5×10⁵ cells/mL. At confluence, a chamber contains ˜150cells; i.e., a concentration of ˜4×10⁷ cells/mL. This concentrationgreatly exceeds the limits of conventional batch cultures. Thus, it isnot surprising that cultures exhibited a strongly inverse correlationbetween the number of cells inoculated into each isolated chamber andthe duration of cell growth, in the absence of the iso-osmotic bathbatch mode (FIG. 11). This underscores the importance of medium exchangeto sustain the continued optimal growth of these cells innanolitre-volume chambers, and the need to progressively increase thefrequency of medium exchange as the number of cells in each cultureincreases. For the single ND13 cell cultures, we found that mediumexchanges at 24, 36, 48, 54, 60, 66 and 72 hours were sufficient toavoid nutrient limitations and the build-up of growth-inhibitingmetabolites, although this feeding pattern can be adjusted based on celltypes, seeding density and required nutrient concentrations.

Example 1.4 Assessment of Growth Heterogeneity by Defined CellPopulations

Time-lapse imaging and automated image analysis was used to generateindividual growth curves for 243 single ND13 cells over a period of 72hours (a sample is shown in FIG. 12 a). After that time, the fastestgrowing clones became multilayered and too large for further tracking byimage analysis. Although the average doubling time for all cells was16.8 hours, we observed substantial heterogeneity in the growthcharacteristics of individual clones. 52% of the input cells either didnot divide or produced progeny that died before the end of theexperiment. This widespread death was offset by the rapid proliferationof other cells that divided as frequently as every 12 hours, but withlarge variability between clones (FIGS. 12 b and 12 c). Such variableclone size distribution was also observed for ND13 cells generatingclones in the 96 well plate cultures (not shown). Furthermore, similardistributions of doubling times were found for both ND13 cells grown inmicrofluidic arrays and multiwell macro plate controls (not shown). Asimilar experiment was conducted using normal primary HSCs. Microfluidicculture of freshly isolated CD45+EPCR+CD48−CD150+ (E-SLAM) adult mousebone marrow cells, which are approximately 50% pure HSCs (Schroeder, T.Cell Stem Cell 1, 479-481 (2007)), over 5 days showed that the kineticsof three successive divisions were comparable to those obtained inmacroscale cultures (Dykstra, B. et al. Proc. Natl. Acad. Sci. USA 103,8185-8190 (2006); Kent, D. G. et al. Blood 112, 560-567 (2008)) (seeFIG. 21).

To further investigate this heterogeneity, we stained ND13 cells for thelineage (lin) markers (Gr-1, Mac-1, and B-220) and compared the clonalgrowth kinetics of single differentiated (lin⁺) and primitive (lin⁻)cells. We opted for lineage staining to characterize cells in thisparticular experiment but antibody staining, enzymatic assays, dyes,RT-qPCR, sequencing, functional assays, or bead capture could also beused to characterize the cells. After staining, cells were introducedinto the device and imaged every 5 minutes for 72 hours. We used theperfusion capabilities to perform a second lineage staining on clones atthe end of the experiment without disturbing colony locations. Thisexperiment showed that most of the lin⁺ cells did not produce colonies(FIG. 13 a), which replicated the failure of lin⁺ cells to form coloniesin 96-well cultures. In contrast, the single lin⁻ cells produced clonesefficiently but of different sizes and phenotypes (FIG. 13 b). Some ofthe lin⁻ cells gave rise to exclusively lin⁺ or lin⁻ clones. In othercases, the clones were of mixed phenotypes (FIG. 13 c). This suggeststhat ND13 cells maintain a lin⁻ clonogenic progenitor population thatcan produce both more of themselves (i.e., lin⁻ cells) as well as moremature non-clonogenic lin⁺ cells. Further support for this model wasobtained by isolating lin⁻ cells by FACS, expanding them in macroscalecultures, and then demonstrating after 12 days that a new lin⁺population had again been produced (FIG. 18).

In a different experiment, unseparated ND13 cells were cultured in themicrofluidic device for 72 hours and then approximately 720 cells from 5chambers (corresponding to 11 starting cell equivalents) recovered (FIG.13 d) and plated into triplicate colony-forming cell assays inmethylcellulose-containing medium. Parallel methylcellulose assays wereset up with the progeny of 11 starting cell equivalents generated instandard control cultures. The number of colonies obtained from eachsource was again similar, further demonstrating the equivalence of themicrofluidic device in supporting ND13 progenitor expansion (FIG. 13 d).

Example 1.5 Expansion of HSCs

To test the suitability of these microfluidic cell culture arrays tosupport HSC self-renewal divisions, we examined the growth of mouse bonemarrow cells transduced with a NUP98-HOXA10homeodomain (NA10hd) fusiongene, which potently stimulates their ability to expand in vitro withoutany signs of leukemic transformation (Ohta, H. et al. ExperimentalHematology 35, 817-830, doi:10.1016/j.exphem.2007.02.012 (2007); andPineault, N. et al. Molecular and Cellular Biology 24, 1907-1917(2004)). To obtain these cells, we first isolated a highly purifiedpopulation of primary HSCs (with a CD45⁺CD48⁻EPCR⁺CD150⁺ phenotype), andthen transduced these cells with a NA10hd-encoding retroviral vector.The transduced cells were then expanded for 11 days in a macroscaleculture. At the end of this period, replicate aliquots were thentransferred either to the microfluidic array or a control macroscalevessel and cultured for an additional 60 hours. The cells from each ofthese latter cultures were then recovered and decreasing fractions ofthe same starting equivalent number injected into groups of 6 mice each.The total number of cells obtained from the chip and the controlmacrocultures were similar (FIG. 14 a). All mice showed similarreconstitution levels by the transplanted cells for >16 weekspost-transplant, indicative of an overall stem cell expansion of morethan 600-fold compared to the stem cell content of the purified cellsinitially transduced (FIG. 14 b). The mice repopulated with cells fromthe microfluidic array also showed reconstitution of both their myeloidand lymphoid compartments (FIG. 15 a). Notably, the cultured NA10 HSCpopulation contained a greater proportion of fast growing cells comparedto the ND13 cells (FIG. 12 b), consistent with the lack of highly maturecells in the NA10 population.

Example 1.6 HSC Response to Temporally Varied SF Stimulation

Previous work has shown that in vitro exposure of HSCs to lowconcentrations of steel factor (SF) (1 ng ml⁻¹) leads to rapid loss ofHSC function, delayed proliferation and increased death compared toculture in higher concentrations (300 ng ml⁻¹) (Kent 2008 supra).However, the reversibility of the effect of low SF concentrations on HSCsurvival and proliferation is not known. To address this questionmicrofluidic system as described herein were used to test how longquiescent adult HSCs could be exposed to a low SF concentration beforerescue by exposure to a high concentration was no longer possible. Anenlarged microfluidic device consisting of 6,144 individual chambers andadditional inlets and flow control valves to enable parallel studieswith many temporally varied conditions was used (FIG. 16). Six differentconditions in which primary mouse HSCs (E-SLAM isolates of adult mousebone marrow) were exposed to 20 ng ml⁻¹ of interleukin-11 (IL-11) pluseither 1 ng ml⁻¹ SF for the first 8, 16, 24 or 48 h followed by 300 ngml⁻¹ SF for the remainder of the experiment, or constant SFconcentrations of 1 ng ml⁻¹ or 300 ng ml⁻¹ for the entire experiment(not shown). The experiment was repeated twice yielding 5 days ofimaging data for a total of 769 single E-SLAM cells cultured in thedevice. By day 5, the fastest growing clones reached confluence, and wecould no longer quantitatively monitor their size. Growth rates of allclones were compared to the results for the constant high SFconcentration. As a control, the same cells were grown in conventionalmacrocultures in 20 ng ml⁻¹ IL-11 plus either 1 ng ml⁻¹ or 300 ng ml⁻¹SF and these yielded the same growth kinetics as in the microfluidicdevice. Compared to the high [SF] condition, a Cox proportional hazardanalysis of the cell survival over time, defined as the fraction ofstarting cells that remained viable or gave rise to clones, showed nosignificant difference (P>0.1) in survival when the cells were rescuedfrom 1 ng ml⁻¹ SF exposure within the first 16 h of culture (Table 1).

TABLE 1 Cox proportional hazard analysis of mouse HSC survival Conditionn Relative risk (95% CI) P value High [SF] (300 ng ml−1) 294 1.00 —  8 hin low [SF] 107 0.82 (0.64-1.06) 0.13 16 h in low [SF] 76 1.03(0.78-1.36) 0.84 24 h in low [SF] 24 1.27 (0.81-1.99) 0.29 48 h in low[SF] 79 1.78 (1.37-2.31) <0.0001 Low [SF] (1 ng ml−1) 189 1.53(1.25-1.86) <0.0001 CI, confidence interval. —, not applicable. Relativerisks and P values were calculated based on the high [SF] condition.

Prolonged initial exposure to 1 ng ml⁻¹ SF led to a rapid decrease inviability between 16 and 24 h, and after that time, the cells could notbe rescued by exposure to 300 ng ml⁻¹ SF (FIG. 6). Most dividing cellscompleted a first mitosis between 24 and 60 h of culture for allconditions, and the SF concentration did not affect the cell divisionkinetics (FIG. 7 and FIG. 8). Analysis of the second division showedcomparable kinetics, with more than 80% of the clones remaining viableafter a first division regardless of the SF concentration to which theyhad been initially exposed. Thus, although a high SF concentrationinfluenced the viability of HSCs as they exited quiescence, it did notdirectly impact the subsequent division kinetics of cells that completea first division.

Microfluidic technology brings the potential of chemical control of theculture medium in combination with single cell imaging to create newopportunities for the high throughput analysis of clonogenic cellresponses to varying extracellular cues. The embodiments describedherein introduce several design features which enable experiments withheterogeneous populations of suspension cells, even those that havestringent medium requirements. Some particular embodiments may includethe incorporation of an enclosed and sterile iso-osmotic reservoir tocontrol unwanted permeation and dehydration effects separated by amembrane from high aspect ratio wells to contain and immobilizenon-adherent test cells during perfusion, and the use of a reverseperfusion strategy or selective aspiration to recover all cells orselected clones.

Using aspects and embodiments described herein, it is demonstratedherein that successful culture of cytokine-dependent hematopoietic cellsis possible with expansion and enhanced HSC function. Maintainingequilibrium with the macroscopic volume of the osmotic bath allows forhigh-throughput microfluidic single cell cultures in volumes that are 4orders of magnitude smaller than conventional macroscale cultures. Asingle cell isolated in a 4 mL chamber is at an effective density of2.5×10⁵/mL, thus making possible the investigation of autocrinesignaling by isolated cells, with the potential of increasing platingefficiency for cell types that might otherwise require conditionedmedium or a high cell density. Co-culture of different cell types atlimiting dilution could further be used to investigate the effect ofcell-cell influences through secreted factors. It is also worth notingthat with sufficient medium exchange, we were able to maintain cellproliferation to densities that resulted in the creation of multiplelayers of cells. This ability to maintain high-density cultures offersnew opportunities for studying the effects of cell concentration on cellbehavior.

Various embodiments also provide flexibility to monitor the clonalgrowth (or other responses) of single non-adherent cells over time inthe presence of dynamic changes in medium conditions by combinedtime-lapse imaging with programmed medium exchanges that do not disturbthe spatial position of each cell or colony. It has been shown, forinstance, that exposure of HSC in vitro to sub-optimal steel factorconcentrations can induce their differentiation within 16 hours evenprior to their entry into the cell cycle (Kent, D. G. et al. Blood 112,560-567, doi:10.1182/blood-2007-10-117820 (2008)). Thus, it is relevantto anticipate that other schedules of growth factor delivery can furthermodulate HSC fate decisions. The fully programmable system for bothperfusion and image acquisition allows automated and dynamic temporaloperation over the entire duration of the culture experiment, therebyproviding a mean to analyze the evolution of clonal cultures in timerather than measuring only end-point outcomes. In addition, the abilityto replace the culture medium is a key feature to avoid nutrientlimitations that occur in longer-term experiments in which even a smallamount of proliferation causes a significant increase in the local cellconcentration. Imaging the cellular contents of 1,600 chambers requiresless than 5 minutes allowing the changes to be monitored at hightemporal resolution. When coupled with emerging image processing toolsfor identifying new morphological phenotypes (Cohen, A. R. et al. NatMeth 7, 213-218 (2010)) and for tracking different cell divisionsidentified by specific markers or fluorescent reporters (Eilken, H. M.et al. Nature 457, 896-900 (2009); and Satyanarayana, S. et al. Journalof Microelectromechanical Systems 14, 392-399 (2005)), the combinedadvantages of high throughput and medium control will now allowpreviously impossible large-scale studies of fate-choices by rare celltypes.

The growth kinetics analysis performed on the ND13 population yieldedfindings that can only be revealed from clonal analyses. The scale ofthe perfusion microfluidic cell culture array described here waspurposefully optimized for the study of small numbers of hematopoieticcells, but can be readily modified to give designs with other features;e.g. more or larger chambers. Situations where only a small fraction ofthe cells are responsible for the long-term maintenance of the overallpopulation are not exclusive to the hematopoietic system. The technologyis highly suitable for adaptation to other cell types/organisms andother applications such as drug-response screens, culture optimization,clone selection, recombinant protein production and cellcharacterization. Various aspects and embodiments described herein arealso ideally suited to controlled experiments investigating theinteraction of two or more cell types. The extended use of microfluidicsystems coupled with live-cell microscopy thus offers great promise formany applications of scientific investigation in biology and medicine.

Example 2 Clonal Cell Growth and Selection Example 2.1 MicrofluidicSecretion Assay and Clonal Expansion

In clonal cultures the accumulation of secreted antibody sufficient fordetection normally requires many days of culture (e.g. 2 weeks),considerably extending the duration of initial cell specificproductivity screens. This analysis is confounded by varied clonalgrowth rates and influenced by evaporation from small volume cultures.To address these challenges we developed a system to analyze within afew hours the productivity of single cells by taking advantage of thefar more rapid product accumulation in nanoliter-volume chambers. Ourmicrofluidic platform also was fabricated using the advantages ofmultilayer soft lithography as described herein. Each of the 1,600chambers in the array was 4.1 nl in volume. The microfluidic deviceincluded micropumps downstream of the array to control loading rates andisolation valves to sequester all of the chambers when needed. In afirst step, we loaded into the array a pool of transfected CHO cellsproducing varied levels of a recombinant monoclonal human IgG1 antibody.Different seeding concentrations were tested and we found that samplesat 2×10⁶ cells ml⁻¹ yielded a high proportion of chambers filled withsingle cells (typically 300-400 out of 1,600), close to the theoreticalmaximum of a Poisson distribution for this stochastic loading (FIG. 22).The high aspect ratio of the chambers sequestered suspension cells bygravity on the bottom of the chambers (FIG. 23, panel a). Cells werethen washed thoroughly to remove antibodies in the medium. Cloningmedium containing polystyrene beads coated with protein A (diameter: 4.9μm) was then introduced into the device and these beads allowed tosettle (FIG. 23, panel b). A medium wash was performed to clear beadsthat had not settled in the chambers (e.g. in the channels betweenchambers), and the isolation valve was then closed for 2 h (FIG. 23,panel c). Following the incubation period, the array was washed withmedium (FIG. 23, panel d) and a solution of labeled detection antibodywas loaded into the device. The chambers were isolated for an additional15 min (FIG. 23, panel e). We tested multiple antibodies and observedthat F(ab′)₂ fragments generally showed lower non-specific binding,consistent with their lack of constant region with affinity for ProteinA (FIG. 24). The Dylight 594-conjugated F(ab′)₂ fragment of rabbitanti-human IgG (H+L) gave the highest signal to noise ratio and wasselected for the assay. The array was washed extensively to remove anyunbound fluorescent antibody (FIG. 23, panel f). A custom algorithm wasthen used to automatically focus on the beads and sets of bright fieldand fluorescent images were acquired from the entire array. After theassay, the isolation valve was left open and the cells were cultured inbatch mode for 4 days (FIG. 23, panel g). High producer clones with goodproliferative capacity were then recovered from the device for furtherexpansion.

Example 2.2 Assessment of Productivity from Single Cells

Custom scripts were developed to automatically segment the beads onbright field images and measure the fluorescence intensity inbead-covered areas (FIG. 25). The mean bead intensity was calculated bydividing the total bead intensity in a chamber by the total projectedarea of the beads. Since beads often merged during segmentation, weestimated the numbers of beads by dividing total bead area by thetheoretical projected area of one bead. Using a concentration of 2 mgbeads ml⁻¹ typically resulted in on average 100-150 beads per chamber.An example of the bead distribution is provided in FIG. 26. We havedeveloped a custom software to assess the accuracy of our image analysisalgorithm and to manually correct bead segmentation errors. Aside from afew outliers, our bead immunocapture algorithm identified the topproducer cells from a population (FIG. 27), thereby demonstrating thepossible rapid automation of the assay. To distinguish the highestproducers from the rest of the population, we needed to ensure that thebeads were below saturation. We generated a curve of the mean beadintensity by making serial dilutions from a known concentration sampleof the secreted IgG1 antibody. The bead saturation occurred at 8 μg ofantibody (mg beads)⁻¹ (FIG. 28), consistent with the manufacturerspecifications. Typical mean intensity values obtained in the beadimmunocapture assay for the top 5% producers cells fell below thesaturation level of the beads.

We first tested whether the bead immunocapture assay had sufficientsensitivity to distinguish producer cells from a pool of CHO cellstransfected to secrete a human IgG1 monoclonal antibody. Since thestochastic loading introduced few cells into the array, we were able tocompare the mean bead intensities of chambers containing single cells tothe intensities of cell-free chambers. Highest producers had mean beadintensities that were 2 orders of magnitude larger than non-producingcells and the detectable producer cell population (32.7% of the cells)was easily distinguished from the low or non-producing population whosechamber intensity distribution matched that of the cell-free chambers(FIG. 29).

Example 2.3 Enhanced Cloning Efficiency

To test whether the microfluidic array could sustain maximal growthrates in clonal cultures, we compared the average growth rates of singlecells in the microfluidic array to the average growth kinetics of singlecells in multiwell plates (culture volume: 200 μl) and shake flaskcultures seeded at 2.5×10⁵ cells ml⁻¹. The cells had reduced growthrates when seeded as single cells in multiwell plates compared to shakeflask cultures. In contrast, clonal cultures in the microfluidic devicehad growth rates comparable to the shake flask cultures (FIG. 30, panela). A single cell in a 4-nl microfluidic chamber is at an effectiveconcentration of ˜2.5×10⁵ cells ml⁻¹, and thus quickly provides aconditioned medium environment as these concentrations do in shake flaskcultures. We then asked whether the microfluidic array could increasethe cloning efficiency due to this higher seeding concentration. We usedthe percentage of clones with more than 8 cells to calculate the cloningefficiency. Clones in multiwell plates on average doubled every 23.2 h,and therefore clones with normal growth are expected to have gonethrough at least 3 divisions after 3 days. Single cells cultured in themicrofluidic array had a significantly higher cloning efficiency thansingle cells cultured in multiwell plates (P value=0.06), most likelydue to medium conditioning effects amplified by the 4 nl volumes (FIG.30, panel b). This enhanced cloning efficiency could allow the recoveryof high-producer clones that would not survive limiting dilution inmultiwell plates.

Example 2.4 Correlation of Surface-Bound mAb with Secretion

Membrane-bound antibody staining has been used as a tool to enrich forhigh producer cells^(13,32). However, there has been contradictingreports as to whether membrane-bound antibody staining is a reliableindicator of the amount of secreted proteins^(13,19,33). Our technologycan conveniently measure membrane-bound and single-cell specificproductivity simultaneously. We observed many instances where secretingcells did not stain for antibodies on their surface (e.g. FIG. 31, panela) and, inversely, where non-secreting cells exhibited surfaceantibodies (e.g. FIG. 31, panel b). We did not see a strong correlation(R²=0.22) between the specific cell productivity and cell intensity(FIG. 31, panel c). While the population with high levels ofsurface-bound antibody appears to contain a greater fraction of highproducer cells, our results show that this technique often excludescells with high productivity and includes cells with low productivity.

Example 2.5 Recovery and Expansion of High-Producer Clones

Clones from the same pool of CHO cells were isolated and then analyzedfor their antibody productivity in the microfluidic array, cultured for5 days and then transferred into 96-well plates. Out of the 308 clones,60 were recovered including the 10% top producers as well as medium, lowand non-producer clones. Of the 60 clones, 95% continued to proliferatein the 96-well plates (e.g. FIG. 32). They were cultured for 9additional days, then transferred into 1 ml of medium in 24-well platesand after 5 days of culture, the supernatant was recovered for titeranalysis. As shown in FIG. 33, panel a, all single cells withoutproductivity in the microfluidic screening were also negative afterrecovery, suggesting that there was no cross-contamination during therecovery process. Also, the microfluidic and multiwell measurementsfollowed similar trends. We further scaled up to batch shake flaskcultures 10 of the top 12 clones identified by the single-cellmicrofluidic assay. Four of those clones had already decreased theirproductivity at the 24-well plate stage (FIG. 33, panel b) and, likewisedid not perform well in the shake flasks (FIG. 34). The six high-rankedclones that had maintained their productivity at the 24-well plate stage(2% of the initial population) gave titers between 200-500 mg ml⁻¹ (FIG.33, panel c) with maximum specific productivities ranging from 6-10 pgcell⁻¹ day⁻¹ (FIG. 33, panel d). The highest single-cell productivityidentified with the microfluidic assay also gave the highest cellspecific productivity in shake flask cultures, demonstrating the abilityof the microfluidic single-cell secretion assay to identify and generatehighly productive cell lines. The recovery, analysis and expansion of 10high-ranked clones from the device led to 6 clones with SPR above 6 pgcell⁻¹ day⁻¹. Our method greatly reduces the burden of screening andsampling large numbers of multiwell plates to find the highestproducers.

Example 2.6 Analysis of Clonal Heterogeneity

We then used our single cell microfluidic assay to determine the cellsecretion profile of our top-ranked clone. As expected, the distributionwas much tighter compared to the cell secretion pool and 97.3% of cellsshowed secretion levels above background (FIG. 35, panel a). The rangeof bead intensity spanned over 3 orders of magnitude, suggestingrelatively high diversity within the population. The standard deviationcorresponded to 68.4% of the mean, a value comparable to what has beenpreviously reported for intraclonal heterogeneity based on intracellularfluorescent reporters (Pilbrough, W. et al. Plos One 4(12), 11 (2009)).Since this clonal population was much more homogeneous than the cellpool, we analyzed the intensity of wells containing more than one cell.There was a linear correlation between the mean bead intensity and thenumber of cell up for up to 3 cells (R²=0.99), after which beads startedto approach saturation (FIG. 35, panel b). This data confirms thequantitative nature of the assay when performed on single cells.

The microfluidic platform described herein provides many advantages forthe selection of high producer clones. Single-cell analysis, wherein onecell is retained in a chamber, allows hundreds of cells to be screenedin a few hours, thereby eliminating the need to expand large numbers ofclones in multiwell plates. This results in considerable economies inculture medium, culture vessels, time and labor. The prototype used inthis study contained 1,600 chambers, enabling the analysis of 300-400single cells per experiment based on the typical seeding density shownin FIG. 22. The design is easily scalable, whereby the number ofchambers could be increased by several folds for populations where moreclones need to be analyzed. Screening of a larger number of cells couldhelp find clones with higher productivities without increasing thenumber of clones to be scaled up. This microfluidic approach differsfrom other single-cell secretion assays by allowing the analysis andculture of cells directly in medium without the need for a semi-solidmatrix to contain the colonies and their secreted product. As describedherein, the system may also be optimized for testing of hematopoieticstem cells to obtain robust growth rates and cellular functionscomparable to large-scale cultures. The sensitivity of CHO cells tomedia conditioning revealed the ability of miniaturization to provide amore adequate culture environment for small numbers of cells. Nanovolumechambers allowed cells to be assayed and cultured at seedingconcentrations comparable to shake flask cultures. The higher cloningefficiency obtained from sequestering cells in small volumes can selectadditional clones that only thrive at high cell concentration. Extendingthe culture for 4-5 days allows a clone to expand to numbers much morelikely to survive when they are transferred to a multi-well plate, inturn increasing the number of high producer clones recovered. Sincecells were analyzed within 2 hours of being retrieved from a shake flaskculture, they are more likely to be in a similar state to suspensioncultures than if they were assayed after multiple days of static culture(e.g. 96-well plate). Together these features bring the rightenvironment to obtain good prediction of the cell specific productivityin batch shake flasks cultures from single-cell measurements. It couldbe possible to further exploit the flexibility of the system byretrieving cells from a fed-batch culture for analysis and assaying themin conditioned media, leading to an even closer match to the conditionstypically used for large-scale mAb production.

Our platform provides similar flexibility and throughput as single-cellsecretion assays based on microengraving methods (Love, J. C. et al.Nature Biotechnology 24(6), 703-707 (2006)), but the integration of thebead immunocapture in an enclosed device has the advantages of allowingfor in situ clonal expansion at non-diluted concentrations. It could bepossible to capture the secreted antibody directly onto ProteinA/G-coated PDMS chambers instead of using bead immunocapture. However,the hydrophobic nature of PDMS provides a low-binding surface, which canbe a desirable feature to maintain cells that have been adapted forsuspension culture. As well, the use of beads in proximity of the cellsconcentrates the signal and possibly leads to better sensitivity. Withmicroengraving, fibronectin coating and attachment periods of up to 6-12h are needed to ensure that single cells are not lost when the glassslide is removed, and then cells are trypsinized for recovery (Park, S.et al. Journal of Biotechnology 156(3), 197-202 (2011)). The high-aspectratio chambers described herein gently capture the clones by gravity,enabling suspension-adapted cell lines to remain on a non-adhesivesurface and to be easily recovered. As the entire process usesintegrated microfluidics, it could easily be fully automated.

The easy coupling of microfluidic devices with time-lapse imaging allowsclonal tracking over time, including the initial verification ofclonality. That colonies arise from single cells can be confirmed byvisual observation. Alternatively, the process could be automated withhigh accuracy using a live/dead stain of the cells. The ease ofautomation of programmable microfluidic systems make this platform wellsuited for industrial applications. For the production of therapeuticmAbs, the detection antibody could be replaced by a labeled recombinantProtein A and hence provide a process devoid of animal components.

Predicting the performance of an entire clone from a single cell within6 hours (for example, between 5 minutes to 6 hrs—for the CHO cellsdescribed in Example 2 the determination was made within 2 hrs) could beinfluenced by significant temporal variations in mAb secretion. Reportshave shown that variations in secretion levels throughout cell cycle aremainly attributed to changes in cell size (Pilbrough, W. et al. Plos One4(12), 11 (2009); and Lloyd, D. R. et al. Cytotechnology 34(1-2), 59-70(2000)), but since those were made on population measurement it is notclear whether the productivity of a single cell changes as it grows.Single-cell protein secretion in yeast has been measured bymicroengraving and no relation was found between productivity and cellcycle stage (Love, K. R. et al. Biotechnology and Bioengineering 106(2),319-325 (2010)). Single-molecule analysis of mRNA transcripts have shownbursts of transcription by CHO and other cell types (Raj, A. et al. PlosBiology 4(10), 1707-1719 (2006); and Raj, A. et al. Nature Methods5(10), 877-879 (2008)), leading to large fluctuations in transcriptexpression. However, simultaneous measurements of transcript and proteinlevels have showed that transcriptional fluctuations are not entirelypropagated to secretion levels (Pilbrough, W. et al. Plos One 4(12), 11(2009)) with the secretion machinery being the limiting factor at hightranscriptional levels (Fann, C. H. et al. Biotechnology andBioengineering 63(4), 464-472 (1999); Schroder, M. & Friedl, PBiotechnology and Bioengineering 53(6), 547-559 (1997)). This suggeststhat even though secretion rates can be influenced by stochasticvariations (Love, K. R. et al. Biotechnology and Bioengineering 106(2),319-325 (2010)), screening results based on single-cell secretion areless prone to be confounded by temporal variations than would bemeasurements obtained from transcriptional analysis at the single-celllevel (e.g. GFP containing expression vectors). The platform describedherein can measure both surface and secreted mAb levels simultaneously.The assay could further be combined with fluorescent reporters genes andproteins to gain a better understanding of the factors regulating themAb or other cell product production at the transcription, translationand secretion levels.

The generation of stable clones is an important aspect of cell lineselection. Generally, the assessment of stability is performed overmultiple passages. A decline in productivity will propagate to adetectable fraction of the population much faster if the initial assayis done on a single cell rather than a bigger colony. Indeed, a fractionof the highly ranked clones exhibited lower productivities in multiwellplates than in the microfluidic assay. These clones can readily beidentified and eliminated at the 24-well plate stage to avoid investingresources and efforts on the scale up of either poor producers orunstable clones. Single-cell analysis is a powerful tool to assess theheterogeneity in cell populations. Performing the same assay on clonescould be used to identify minor fractions of clonal population that havereduced mAb production rates before a decrease in titers could bedetected. Analysis of intracellular mAb using FACS is used by some as atool to identify unstable clones (Dorai, H. et al. Biotechnology andBioengineering 109(4), 1016-1030 (2011)). Our platform could enablesecretion analysis on single cells from clonal population to obtain anearly assessment of clone homogeneity and detect signs of instability.

There is an increasing need in the industry to accelerate thedevelopment of mAbs and other cell products. The throughput,sensitivity, flexibility and ease of automation of microfluidicsingle-cell analysis systems bring new tools to reach this goal. Cellline selection is only one example from a plethora of immunologicalapplications that could benefit from the combination of clonal cellculture and high-throughput secretion screens. These include hybridomageneration, isolation of rare activated T cells, selection of newantibodies from primary cells or directed evolution in mammalian cells.With the ability to obtain robust and, as shown in this work, moreefficient culture conditions in small volumes, microfluidic devices havepotential to becoming vessels of choice to search for rare cells inheterogeneous populations.

1. A method, the method comprising: (a) retaining a cell at a retainingposition within a microfluidic chamber having a chamber volume; (b)flowing a perfusing fluid through the microfluidic chamber, wherein theperfusing fluid enters the chamber through an inlet at an inlet positionand exits the chamber through an outlet at an outlet position; and (c)measuring a cell product produced by the cell within the microfluidicchamber; wherein the perfusing fluid has a greater velocity laminar flowadjacent the inlet and outlet positions than at the retaining position,and wherein a first region of the chamber is spaced apart from theretaining position, wherein the first region is interposed directlybetween the inlet and outlet positions.
 2. The method of claim 1,wherein measuring the cell product may be selected from one or more of:lineage staining; cell-surface protein staining; antibody staining;enzymatic assay; RT-PCR analysis; PCR analysis; sequencing; functionalassay; and bead capture assay to characterize the cells.
 3. A method,the method comprising: (a) retaining a cell at a retaining positionwithin a microfluidic chamber; (b) flowing a perfusion fluid into themicrofluidic chamber through an inlet; (c) flowing the perfusion fluidout of the microfluidic chamber through an outlet wherein the outlet ispositioned such that gravitational forces acting on the cell to keep itat or near the retaining position exceed hydrodynamic forces acting onthe cell to move it toward the outlet; and (d) measuring a cell productproduced by the cell within the microfluidic chamber.
 4. The method ofclaim 3, wherein measuring the cell product may be selected from one ormore of: lineage staining; cell-surface protein staining; antibodystaining; enzymatic assay; RT-PCR analysis; PCR analysis; sequencing;functional assay; and bead capture assay to characterize the cells.
 5. Amethod, the method comprising: a. retaining a cell in a volume ofperfusion fluid, wherein the volume is less than about 10 nL; b. placingthe volume of perfusion fluid in fluid communication with a reservoirfluid, wherein the reservoir fluid has a volume greater than the volumeof perfusion fluid; and c. measuring a cell product produced by the cellwithin the perfusion fluid.
 6. The method of claim 5, wherein measuringthe cell product may be selected from one or more of: lineage staining;cell-surface protein staining; antibody staining; enzymatic assay;RT-PCR analysis; PCR analysis; sequencing; functional assay; and beadcapture assay to characterize the cells.
 7. The method of claim 2,further comprising selecting cell clones based on cell characteristics.8. The method of claim 7, wherein cell characteristics are selected fromone or more of: quantity of secreted product, quality of secretedproduct, proliferation, morphology, gene expression, fluorescentreporter, surface proteins, genealogical pedigree, viability, apoptosis,autophagy, metabolism, clone homogeneity, and clone heterogeneity.
 9. Amicrofluidic device for perfusing a cell with perfusion fluid, thedevice comprising: a chamber, having: (i) at least one inlet; (ii) atleast one outlet; and (iii) a cell retainer; wherein the inlet and theoutlet are in fluid communication with the cell retainer, and whereinthe outlet is positioned such that, when the device is being perfused,gravitational forces acting on the cell to keep it at or near theretainer exceed hydrodynamic forces acting on the cell to move it towardthe outlet.
 10. A microfluidic device for perfusing a cell withperfusion fluid, the device comprising: a chamber, having: (i) at leastone inlet; (ii) at least one outlet; and (iii) a cell retainer; whereinthe inlet and the outlet are in fluid communication with the cellretainer; and wherein a first region of the chamber is interposeddirectly between the inlet and outlet positions and is spaced apart fromthe cell retainer.
 11. The microfluidic device of claim 9, furthercomprising a perfusion fluid comprising one or more of the following: animmunostaining agent; an enzymatic reagent; a dye; and a functionalizedbead.
 12. The microfluidic device of claim 11, wherein (a) theimmunostaining agent is selected from one or more of: monoclonalantibodies; polyclonal antibodies; fluorophores; and blocking solution;(b) the enzymatic reagent is selected from one or more of: horseradishperoxidase; and luminol; (c) the dye is selected from one or more of:rhodamine; fluorescein; calcein; Hoescht; Trypan blue; propidium iodide;and Giemsa solution; (d) the functionalized bead is selected from one ormore of: magnetic beads; polymer beads; protein A-coated beads; andprotein G-coated beads.
 13. A method of culturing a cell and measuringcell products within a microfluidic chamber, the method comprising: (a)retaining the cell within the microfluidic chamber at a concentration of≧10,000 cells per mL; (b) flowing a perfusing fluid through themicrofluidic chamber; and (c) measuring a cell product produced by thecell within the microfluidic chamber.
 14. The method of claim 13,wherein the cell product is secreted by the cell.
 15. The method ofclaim 13, wherein the cell product is quantified directly or indirectlyby an intracellular fluorescent reporter.
 16. The method of claim 13,wherein the measuring of the cell product is selected from one or moreof the following: antibody staining; enzymatic assaying; functionalassaying, surface capturing, or bead capturing.
 17. The method of claim13, wherein the cell or clone is maintained under culture conditionsthat allow subsequent clonal expansion.
 18. The method of claim 13,wherein the cell within the microfluidic chamber is at a concentrationselected from one of the following: ≧20,000 cells per mL; ≧30,000 cellsper mL; ≧40,000 cells per mL; ≧50,000 cells per mL; ≧60,000 cells permL; ≧70,000 cells per mL; ≧80,000 cells per mL; ≧90,000 cells per mL;≧100,000 cells per mL; ≧125,000 cells per mL; and ≧250,000 cells per mL.19.-28. (canceled)
 29. The method of claim 13, wherein measuring thesecretion product occurs between 5 minutes and 6 hours from theretaining of the cell within the microfluidic chamber.
 30. The method ofclaim 4, further comprising selecting cell clones based on cellcharacteristics.
 31. The method of claim 6, further comprising selectingcell clones based on cell characteristics.
 32. The microfluidic deviceof claim 10, further comprising a perfusion fluid comprising one or moreof the following: an immunostaining agent; an enzymatic reagent; a dye;and a functionalized bead.